Genetic suppression and replacement

ABSTRACT

The invention relates to gene suppression and replacement. In particular, the invention relates to enhanced expression of suppression agents for suppressing gene expression in a cell and in vivo and replacement nucleic acids that are not inhibited by the suppression agent. Regulatory elements are included in expression vectors to optimize expression of the suppression agent and/or replacement nucleic acid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application under 35 U.S.C. §120 of acurrently pending U.S. application Ser. No. 12/595,080 filed on Mar. 3,2012 which is a 371 National Phase Entry Application of InternationalApplication No. PCT/GB2008/001310 filed Apr. 14, 2008, which designatedthe U.S., and which claims the benefit under 35 U.S.C. 119(e) of U.S.Provisional No. 60/923,067 filed Apr. 12, 2007, the contents of whichare incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 3, 2010, isnamed20100303_ReplacementSequenceListing_TextFile_(—)740789_(—)066400_US.txtand is 127,586 bytes in size.

FIELD OF THE INVENTION

The invention relates to mutation independent suppression andreplacement of disease-causing mutant genes.

BACKGROUND OF THE INVENTION

Many mutation-based diseases are more genetically diverse than can bepredicted from clinical presentation. Some mutation-based diseases areMendelian and involve the inheritance of a single mutant gene, othersare polygenic or multifactorial and involve multiple genetic insults. Inthe case of some Mendelian disorders, many different mutations withinthe same gene can give rise to, or can predispose an individual to, adisease. Similarly, for some multifactorial disorders, many differentmutations within one or more genes can predispose an individual to adisease or can act in an additive manner with other genetic andenvironmental influences to give rise to a disease. This mutationalheterogeneity underlying the molecular etiologies of many diseasesrepresents a significant barrier to the development of therapies forsuch diseases. Moreover, genetic strategies for suppressing andreplacing a mutant protein face many challenges with regard to theeffectiveness of the machinery used to deliver and regulate theexpression of the suppressor and replacement nucleic acids in vivo.Therefore, a need exists for effective mutation-independent therapeuticsthat achieve effective suppression and replacement.

SUMMARY OF THE INVENTION

The invention relates to gene suppression and replacement. Inparticular, the invention relates to enhanced expression of suppressionagents for suppressing gene expression in a cell and in vivo and ofreplacement nucleic acids that are not inhibited and/or are partiallyinhibited by the suppression agent. Expression vectors used to expressthe suppression agent(s) and replacement nucleic acids compriseregulatory elements to optimize expression of the suppression agent(s)and or replacement nucleic acids.

The invention embodies use of replacement genes using sequences toenhance expression of replacement genes from viral and or non-viralvectors. In a further aspect the invection relates to enhancedexpression of suppression agent(s) and or replacement genes from viralor and non-viral vectors. In a further embodiment the invention relatesto enhanced expression of suppression agent(s) and or replacement genesand or genes encoding neurotrophic factors from viral and or non-viralvectors.

In one aspect the invention relates to use of conserved sequences fromretinal genes to enhance expression of suppression agent(s) and orreplacement genes and or genes encoding neurotrophic factors. The use ofsuch conserved sequences has been found to result in surprisinglyefficient expression. In a particular aspect the invention relates touse of conserved sequences from retinal genes to enhance expression ofsuppression agent(s) and or replacement genes and or genes encodingneurotrophic factors from adeno associated virus (AAV) vectors. Inanother aspect the invention provides vectors for expression ofsuppression agent(s) and or replacement gene(s) and or genes encodingneurotrophic factors using regulatory sequences from retinal gene(s) andor non-retinal gene(s) and or ubiquitously expressing genes to enhanceexpression from vectors.

In one aspect, the invention provides vectors for expressing asuppression agent for a disease causing gene and/or a replacementnucleic acid that is not recognized or is partially recognized by thesuppression agent.

In an embodiment, the vector comprises an enhancer sequence, such as,for example, a sequence of SEQ ID NOs: 402-413 or functional variants orequivalents thereof. In another embodiment, the vector comprises atleast one regulatory element selected from the group consisting of apromoter, a stuffer, an insulator, a silencer, an intron sequence, apost translational regulatory element, a polyadenylation site, and atranscription factor binding site.

In another embodiment, the vector comprises at least one of conservedregions A through I from the rhodopsin gene, as represented by SEQ IDNOs: 92-99, or functional variant or equivalent thereof. In anotherembodiment, the vector comprises at least one transcription factorbinding site sequence selected from the group consisting of SEQ ID NOs:100-401, or functional variant or equivalent thereof.

The suppression agent may be a nucleic acid, protein, amino acid(s),antibody, aptamer, or any such agent that can bind to and inhibit a DNA,RNA, or protein. In an embodiment, the suppression agent is a siRNAselected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35-67, 75, 77, 79, 81, 83, 85,and 414-421 or functional variant or equivalent thereof.

The replacement nucleic acid is not recognized or is recognizedpartially by the suppression effector, because its sequence has beenaltered such that it cannot bind or binds less efficiently to thesuppression agent but still encodes a normal or enhanced gene product.In an embodiment, the replacement nucleic acid is a siRNA selected fromthe group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 68, 76, 78, 80, 82, 84, and 86, orfunctional variant or equivalent thereof.

In an embodiment, the invention provides vectors, such as viral vectors,that comprise a suppression agent and/or a replacement nucleic acid. Forexample, the vector comprises at least one suppression agent nucleotidesequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35-67, 75, 77, 79,81, 83, and 85, or functional variant or equivalent thereof, and atleast one replacement nucleic acid nucleotide sequence selected from thegroup consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 68, 76, 78, 80, 82, 84, and 86, or functionalvariant or equivalent thereof.

In another aspect, the invention provides therapeutic compositionscomprising at least one vector comprising at least one suppression agentnucleotide sequence selected from the group consisting of SEQ ID NOs: 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35-67, 75,77, 79, 81, 83, 85 and 414-421 or functional variant or equivalentthereof, and at least one replacement nucleic acid nucleotide sequenceselected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 68, 76, 78, 80, 82, 84, and86, or functional variant or equivalent thereof. In an embodiment, thevector of the therapeutic composition further comprises a regulatoryelement selected from the group consisting of an enhancer, a promoter, astuffer, an insulator, a silencer, an antirepressor, an intron sequence,a post translational regulatory element, a polyadenylation signal (e.g.minimal poly A), a conserved region A through I, and a transcriptionfactor binding site.

In another aspect the invention provides suppression and replacement inconjunction with provision of a gene encoding aneurotrophic/neuroprotective factor(s).

In another aspect, the invention provides cells comprising the nucleicacids and vectors of the invention.

In another aspect, the invention provides transgenic animals comprisingthe nucleic acids and vectors of the invention.

In yet another aspect, the invention provides methods of suppressing theexpression of a mutant gene and replacing expression of the mutant genewith a replacement nucleic acid, the method comprising administering toa mammal a therapeutic composition of the invention.

In yet another aspect, the invention provides methods of suppressing theexpression of a mutant gene and replacing expression of the mutant genewith a replacement nucleic acid in conjunction with a gene encoding aneurotrophic/neuropeotective factor(s), the method comprisingadministering to a mammal a therapeutic composition of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of the presentinvention, as well as the invention itself, will be more fullyunderstood from the following description of preferred embodiments whenread together with the accompanying drawings, in which:

FIG. 1 illustrates RHO suppression and replacement constructs. FIG. 1Ais a diagrammatic representation of a RHO suppressor-EGFP constructshBB-EGFP (shQ1-EGFP and shNT-EGFP have the same format). shRNAs wereexpressed from the H1 promoter and EGFP from the CMV immediate earlypromoter. The SV40 polyadenylation signal was located at the 3′ end ofthe EGFP gene. FIG. 1B illustrates a two component suppression andreplacement construct shBB-rBB (shQ1-rQ1 and shNT-rBB have the sameformat). Suppressors were expressed from the H1 promoter and replacementRHO cDNAs from a 1.7 kb mouse rhodopsin promoter (rhoP). Polyadenylationsignals of the RHO gene were included in the 1829 bp fragment. HGH int:human growth hormone intron. For tissue culture and retinal explantexperiments these constructs were maintained in pEGFP-1 (A) or aCMV-promoterless derivative of pcDNA-3.1- (B) and for in vivoexperiments in the AAV vector. Restriction enzyme sites used for cloningare indicated. Promoters were separated by spacer DNA fragments. Numbersindicate molecular sizes (bp) and arrows indicate direction oftranscription.

FIG. 2 illustrates RHO suppression in HeLa cells. HeLa cells weretransiently co-transfected three times in triplicate with wild type RHOand RHO-targeting siRNAs (siB, siBB, siC, siCC, siQ1 or siQ2) or controlsiRNAs (siEGFP or siNT). Following transfection, RHO mRNA and proteinlevels were evaluated by real time RT-PCR (A), ELISA (A) and Alexa Fluor568-labeled immunocytochemistry (B). Cell nuclei were counterstainedwith DAPI. Error bars represent SD values.

FIG. 3 illustrates replacement of RHO expression in conjunction withsuppression in HeLa cells. Replacement RHO sequences were generated withaltered degenerate nucleotides at siRNA target sites. HeLa cells weretransiently co-transfected three times in triplicate with a replacementRHO expression vector (rBB, rCC or rQ1) and a RHO-targeting siRNA (siBB,siCC or siQ1) or a non-targeting siRNA (siNT). Replacement RHO mRNAlevels were evaluated by real time RT-PCR. Error bars represent SDvalues.

FIG. 4 illustrates RHO suppression in retinal explants. Mouse retinas(n=6), dissected from newborn NHR+/− rho−/− pups (transgenic miceexpressing a human rhodopsin transgene NHR on a mouse rhodopsin knockoutbackground rho−/−), were electroporated with a construct co-expressing ashRNA targeting RHO or a non-targeting shRNA and EGFP (shBB-EGFP,shQ1-EGFP or shNT-EGFP). Negative control explants were notelectroporated. Two week organotypic cultures were dissociated withtrypsin and FACS analysed. Red and blue dots (right and left populationsrespectively in each of A1 and A2) represent gated and ungatedpopulations of dissociated explants. Scatterplots of forward- (FSC)versus side-scatter (SSC) and histograms of EGFP fluorescence of thegated population of non-electroporated (A1 and A2, EGFP-negative) andelectroporated (A3 and A4, EGFP-positive) retinas are given. The barchart indicates RHO mRNA levels in retinal explant cells expressingsNT-EGFP, sBB-EGFP and sQ1-EGFP, quantified by real time RT-PCR. Errorbars represent SD values.

FIG. 5 illustrates RHO suppression in photoreceptor cells in vivo. Adulttransgenic NHR+/− rho−/− mice were subretinally injected with 3 μl2×10¹² vp/ml AAV co-expressing a RHO-targeting or non-targeting shRNAand EGFP (AAV-shBB-EGFP or AAV-shNT-EGFP). Retinas were analysed twoweeks post-injection. Expression of the 21 nucleotide (nt) shRNA BB,detected by RNase protection in two transduced retinas, is depicted inlanes L1 and L2 (A). RHO RNA probes were labelled with P³²-γATP andprotected RNA separated on 15% denaturing acrylamide gels (A). M: sizemarker indicates 10, 20 and 30 nt. Bars represent RHO mRNA levels inFACS sorted cells from dissociated retinas (n=6) transduced with eitherAAV-shBB-EGFP or AAV-shNT-EGFP (B). Suppression levels were determinedby real time RT-PCR. Error bars represent SD values. Rhodopsinimmunocytochemistry (Cy3-labeled) and EGFP protein expression in cellsfrom dissociated retinas, transduced with either AAV-shBB-EGFP (arrows)or AAV-shNT-EGFP (arrow heads), are depicted (C). Cell nuclei werecounterstained with DAPI.

FIG. 6A-D illustrates retinal histology and ERG analysis of RHO-M mouse.Two month old rho+/+(wild type), rho+/−, NHR+/− rho−/− and RHO-M+/−rho−/− mice were analysed by retinal histology and ERG (n=8). A, B andC: rhodopsin immunocytochemistry (Cy3) showing similar rod outer segment(ROS) labelling in rho+/+, NHR+/− rho−/− and RHO-M+/− rho−/− retinasrespectively. Nuclear layers were stained with DAPI. D: representativerod-isolated ERG responses. ONL: outer nuclear layer. INL: inner nuclearlayer. GCL: ganglion cell layer.

FIG. 6E illustrates RNAi-mediated suppression of human rhodopsin inRHO-M mice. RHO-M mice were subretinally injected with AAV2/5 vectorscarrying an shRNA-based suppressor and an EGFP reporter gene. Mice weresacrificed 14 days post-injection, retinas taken and retinal cellsdissociated as in Palfi et al. 2006. RNAi-mediated suppression wasevaluated using real-time RT-PCR assays. Retinal cells transduced withAAV-shBB-EGFP, AAV-shCC-EGFP and AAV-shQ1-EGFP vs AAV-shNT-EGFP wereFACS sorted from adult RhoM mouse retinas, 14 days post subretinalinjection. Of note is that AAV-shCC-EGFP suppresses RHO less in RHO-Mmice due to the presence of a 2 bp mismatch in the human rhodopsintransgenic in RHO-M animals. Levels of rhodopsin expression were shCC:59.73%; shBB: 8.77%; shQ1: 20.6% when compared to the non-targetingcontrol shNT which was set at 100% expression.

FIG. 6F illustrates depression of the ERG response in RHO-M eyes thathave received AAV-shBB-EGFP or AAV-shQ1-EGFP when compared to eyessubretinally injected with AAV-shNT-EGFP. The top tracing in each panelrepresents the right eye which received the targeting AAV-shRNA vectorand the bottom tracing in each panel represents the left eye whichreceived the control non-targeting AAV-shNT vector. In contrast noreduction/depression of the ERG was observed in RHO-M mice subretinallyinjected with AAV-shCC-EGFP vector.

FIG. 7 illustrates the expression of replacement RHO in vivo. Ten dayold rho−/− mice were subretinally injected with a 1:1 mixture of 2 μl12×10¹² vp/ml of two AAV vectors, AAV-EGFP (also termed AAV-CMV-EGFP)and AAV-shBB-rBB (also termed AAV-BB8). Rhodopsin, EGFP protein andnuclei were detected by Cy3-labeled immunocytochemistry, nativefluorescence and nuclear DAPI staining respectively. Low magnificationimages show a cross section of a whole injected eye with arrowheadsindicating the transduced area (A and B). High magnification laserscanning micrographs show transduced (C and D) and non-transduced (E andF) areas. INL: inner nuclear layer. GCL: ganglion cell layer. ROS: rodouter segments. ONL: outer nuclear layer. FIG. 7 provides evidence ofrhodopsin protein expression from replacement genes in retinal sectionsobtained from rho−/− mice subretinally injected with AAV2/5 suppressionand replacement vectors.

FIG. 8 illustrates the histology of AAV-transduced Pro23H is retinas.Newborn Pro23His+/− rho+/− mice were subretinally injected with 1 μl2×10¹² vp/ml AAV-shBB-rBB or AAV-EGFP (n=6). Ten days post-transductioneyes were processed for semi-thin sectioning and stained with toluidineblue. Approximately 40 measurements in three layers per eye of outernuclear layer (ONL) thickness (μm) were taken. A: bars represent ONLthickness, of the central meridian of the eye, of the lowest and highest15% values (p<0.01). B and C: representative images of AAV-shBB-rBB- andAAV-EGFP-(control) injected sections corresponding to highest ONLthickness values. Yellow arrows indicate ONL thickness. INL: innernuclear layer. GCL: ganglion cell layer. Error bars represent SD values.

FIG. 9 illustrates suppression and/or replacement constructs used togenerate recombinant AAV2/5 viruses using the procedures provided inExample 1. RHO suppression and or replacement constructs, pAAV-BB8,pAAV-BB9, pAAV-BB10, pAAV-BB11, pAAV-BB12, pAAV-BB13, pAAV-BB18,pAAV-BB26/Q26, pAAV-BB16, pAAV-BB24 and pAAV-BB27. Illustrations of somecontrol constructs are also provided (pAAV-rho-EGFP and pAAV-CMV-EGFP).Suppression constructs with EGFP reporter genes are also provided(pAAV-shBB-EGFP, pAAV-shQ1-EGFP, pAAV-shCC-EGFP). Suppressors wereexpressed from the H1 promoter and replacement RHO cDNAs fromdifferently sized mouse rhodopsin promoter sequences. HGH int: humangrowth hormone intron. CRX-NRL indicates enhancer element SEQ ID NO: 94.Restriction enzyme sites used for cloning are indicated. Promoters wereseparated by spacer DNA fragments. Numbers indicate molecular sizes (bp)and arrows indicate direction of transcription. Notably, any combinationof the elements and conserved regions outlined and indeed other elementsthat can modulate gene expression could be used in the invention toexert control over expression of suppression and or replacementcomponents.

FIG. 10 illustrates a comparison of levels of expression from the Rho-Mtransgene versus that obtained from the suppression and replacementconstructs in AAV2/5 and represented in FIG. 9, using RNAse protection.FIG. 10 illustrates that the suppression and replacement constructs (seeFIG. 9) engineered into AAV2/5, AAV-BB8, AAV-BB10, AAV-BB11, AAV-BB12,AAV-BB13 and AAV-BB16 express the human rhodopsin replacement gene inRNA extracted from 129 wild type mice subretinally injected withsuppression and or replacement constructs. (Lanes with material frommouse eyes injected with AAV-BB8 are indicated by BB8, AAV-BB10 by BB10,AAV-BB11 by BB11 etc. The plasmid constructs used to generate AAVvectors are written in the format pAAV as presented in FIG. 9). BB8,BB10 and BB11 express rhodopsin at lower levels than BB12, BB13 andBB16.

FIG. 11 provides a comparative analysis of rhodopsin expression fromrAAV2/5 suppression and replacement vectors using real time RT-PCR. FIG.11 illustrates replacement rhodopsin expression levels in RNA extractedfrom 129 wild type mice subretinally injected with suppression and/orreplacement constructs. Expression levels were also determined in Rho-Mtransgenic mice which express a rhodopsin replacement construct rCC anddisplay normal retinal function. Suppression and replacement constructsBB12, BB13, BB16 and BB18 express approximately in the same order ofmagnitude as levels of replacement rhodopsin transcript in Rho-M mice,indicating that enhanced replacement constructs with enhancer elementsand conserved regions may express sufficient levels of rhodopsin tosustain a functional retina in vivo. (Lanes with material from mouseeyes injected with AAV-BB8 are indicated by BB8, AAV-BB10 by BB10,AAV-BB11 by BB11 etc.)

FIG. 12 illustrates retinal histology of adult wild type retinas weresubretinally injected with 2 ul of 2×10¹² particle/ml of differentreplacement-RHO AAV vectors (see FIG. 9). Two weeks post-injectiontransduced eyes were removed, fixed in 4% paraformaldehyde andcryosectioned (12 um). Subsequently, sections were stained with humanspecific anti-RHO antibody to visualize expression of replacement-RHOusing Cy3 label (red) on the secondary antibody; cell nuclei werecounterstained with DAPI (blue). A: AAV-BB8, B: AAV-BB13, C: AAV-BB24,D: AAV-Q8, E: AAV-Q26, F: retina from uninjected RhoM transgenic mouseexpressing RHO (positive control). Sections indicate different levels ofRHO expression in the sections. OS: photoreceptor outer segments; IS:photoreceptor inner segments; ONL: outer nuclear layer; INL: innernuclear layer; GCL: ganglion cell layer.

FIG. 13 illustrates retinal histology of adult NHR transgenic mice on arho−/− background, therefore expressing normal human RHO but not mouserho. These mice were transduced by subretinal injection of 2 ul of2×10¹² particle/ml of AAV-shQ1-EGFP (A) or AAV-shNT-EGFP (B). Two weeksafter injection, eyes were removed, fixed in 4% paraformaldehyde andcryosectioned AAV-shQ1-EGFP expresses shRNA-Q1, which targets RHO, whileAAV-shNT-EGFP expresses a non-targeting shRNA (FIG. 9 illustratesexemplary constructs). Both constructs express EGFP allowing trackingthe transduced cell populations (green). Sections were counterstainedDAPI (blue) to label position of the nuclear layers. A significantreduction in the photoreceptor cell number in the transduced part of theouter nuclear layer is apparent in the AAV-shQ1-EGFP injected (A)retinas compared to those of injected with AAV-shNT-EGFP (B). IS:photoreceptor inner segments; ONL: outer nuclear layer; INL: innernuclear layer; GCL: ganglion cell layer.

FIG. 14A-C illustrates retinal histology of adult RHO-347 transgenicmice carrying a dominant RHO mutation on a mouse rho+/+ backgroundcausing retinal degeneration were subretinally injected with 2 ul of2×10¹² particle/ml of AAV-shNT-EGFP (A) or AAV-shQ1-EGFP (B) vectors.Two weeks post-injection transduced eyes were removed, fixed in 4%paraformaldehyde and cryosectioned (12 um). AAV-shQ1-EGFP expressesshRNA-Q1-EGFP, which targets RHO, while AAV-shNT-EGFP expresses anon-targeting shRNA. Both constructs express EGFP allowing tracking ofthe transduced part of the retina (green). Sections were counterstainedwith DAPI (blue) to indicate positions of the nuclear layers. Asignificant reduction of the photoreceptor cell numbers in thetransduced part of the outer nuclear layer in the AAV-shNT-EGFP injectedor the uninjected (C) retinas are apparent due to the degenerativeeffects of RHO-347 transgene. A significantly preserved outer nuclearlayer is detected in the AAV-shQ1-EGFP transduced retinas, whereshRNA-Q1-EGFP effectively suppresses the RHO-347 transcript thereforereducing retinal degeneration. Note, that mouse rho (expressed in theseretinas) is refractory to suppression by shRNA-Q1-EGFP due to thepresence of nucleotide changes at the target site for Q1 siRNAsuppression. Suppression and replacement using the degeneracy of thegenetic code provided therapeutic benefit at a histological level. FIG.14D provides evidence of an improvement in the electroretinogram (ERG)in RHO-347 eyes treated with AAV-shQ1-EGFP versus eyes treated withAAV-shNT-EGFP. FIG. 14D provides a representative maximum ERG responseof a RHO-347 mouse, containing a human rhodopsin transgene with amutation at codon 347, subretinally injected with AAV2/5 constructs.This RHO-347 mouse normally displays a phenotype similar to autosomaldominant RP. The top panel in FIG. 14D is the response of the right eye,which received an injection of AAV-shQ1, a AAV2/5 vector containingsuppressor siRNA Q1 driven by an H1 promoter (shQ1) and a CMV-drivenEGFP gene. The left eye received an AAV-shNT, a AAV2/5 containing anon-targeting (control) siRNA driven by an H1 promoter (shNT) and aCMV-driven EGFP gene. As can be seen in FIG. 14D, the maximum responseis significantly greater in the treated right eye than in the controlleft eye, indicating that suppression of the mutant rhodopsin transgeneleads to some rescue at the ERG level.

FIG. 15 illustrates exemplary constructs utilising chromatin openingelements to optimise expression are presented. Components utilised toenhance expression may be cloned into vectors such as AAV vectors.Elements to optimise expression of a given gene may be combined withother promoter elements such as the rhodopsin promoter and/or enhancersequences or alternatively sequences that modulate chromatin structuresand drive gene expression may be utilised alone to facilitateoptimisation of expression of a target gene.

FIG. 16 shows sequences of exemplary elements that can facilitatemodulation of chromatin structures.

FIG. 17 shows nucleotide and amino acid sequences of a number ofexemplary neurotrophic factors.

FIG. 18 illustrates exemplary suppression and replacement constructscontaining other genetic elements which are beneficial for photoreceptorcell survival. In the example pAAV-BB18 has been combined withneurotrophic factor GDNF, driven by a small UCOE (chromatin openingelement. A Thrasher, Abstract 36, British Society for Gene Therapy5^(th) Annual Conference 2008) promoter). Other neurotrophic factorssuch as, for example, Neurturin may also be used in combination with anyof the suppression and replacement constructs described. In addition,other beneficial genes, other than neurotrophic factors may also becombined with suppression and replacement constructs such as forexample, a second suppression element, a second replacement element,VEGF and others. In example A, the additional element, in this case GDNFis co-located with the suppression and replacement construct within thetwo AAV inverted terminal repeat sequences, ITR1 and ITR2. In the secondexample, B, the GDNF gene and its promoter are not co-located with thesuppression and replacement elements within ITR1 and ITR2, but arelocated within the backbone of the plasmid used to generate AAV. Since asmall proportion of the backbone is packaged during AAV production, thiswould result in a mixed population of AAVs with the majority containingthe suppression and replacement elements and a minority the GDNFelements. In this case, other beneficial genes, other than neurotrophicfactors may also be combined with suppression and replacement constructssuch as for example, a second suppression element, a second replacementelement, VEGF and others.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention utilises efficient gene suppression in conjunctionwith gene replacement to overcome the challenge of mutationalheterogeneity. The suppression agent does not necessarily target amutation (although it can encompass the site of a mutation), but israther mutation independent. Suppression can involve one or both allelesof an endogenous gene. In conjunction with suppression, a replacementgene is provided that has been modified such that the replacement geneis refractory or partially refractory to suppression. The invention usesthe degeneracy of the genetic code to modify the replacement gene.Alteration of “wobble” bases makes it possible for replacement nucleicacids to escape suppression at least in part, but does not change theprotein product expressed from the replacement nucleic acids.Alternatively, replacement genes are modified in such a way that theyencode altered amino acids but still encode a functional or partiallyfunctional protein that does not lead to pathology (e.g., because theamino acid changes are silent mutations or polymorphisms). Replacementhas been demonstrated using rhodop sin nucleic acids, however, othergenes or combinations of genes can be made and used in the practice ofthe invention. In particular, the invention relates to modulating andoptimizing the expression levels of the suppression agents and/orreplacement nucleic acids using one or more of the untranslated regions(UTRs) of a gene, intronic sequences, the degeneracy of the genetic codeand/or polymorphisms to alter the sequence of replacement nucleic acidssuch that they are refractory or partially refractory to suppression.

In one aspect, the invention provides methods for preparing and using asuppression agent and replacement nucleic acid. The suppression agentbinds to a coding region of a mature RNA or DNA encoding a mutant alleleand inhibits expression of the mutant allele. The replacement nucleicacid encodes a wild-type or non-disease causing allele and comprises atleast one degenerate/wobble nucleotide that is altered so that thereplacement nucleic acid is not suppressed, or is only partiallysuppressed, by the suppression of one or both alleles of a gene.

The invention provides for replacement genes using sequences to enhanceexpression of replacement genes from viral and or non-viral vectors. Inparticular the invention relates to enhanced expression of suppressionagent(s) and or replacement genes from viral or and non-viral vectors.The invention relates to use of conserved sequences from retinal genesto enhance expression of suppression agent(s) and or replacement genes.In a particular aspect the invention relates to use of conservedsequences from retinal genes to enhance expression of suppressionagent(s) and or replacement genes from adeno associated virus (AAV)vectors. In another aspect the invention provides vectors for expressionof suppression agent(s) and or replacement gene(s) using regulatorysequences from retinal gene(s) and or non-retinal gene(s) and orubiquitously expressing genes such as those provided in the Tables belowto enhance expression from viral and non-viral vectors.

In another aspect, the invention provides a composition comprising asuppression agent that binds to the coding region of a mature and/orimmature RNA or DNA encoding a mutant allele to inhibit expression ofthe mutant allele and a replacement nucleic acid that encodes awild-type or non-disease causing allele and comprises at least onedegenerate/wobble nucleotide that is altered so that the replacementnucleic acid is not suppressed, or is only partially suppressed, by thesuppression agent.

In yet another aspect, the invention provides a kit comprising asuppression agent that suppresses the expression of a mature and orimmature RNA or DNA encoding a mutant allele and a replacement nucleicacid that encodes a wild-type or non-disease causing allele that is notsuppressed, or is only partially suppressed, by the suppression agentand differs from the mutant allele in at least one degenerate/wobblenucleotide.

Suppression is achieved using a wide variety of molecular tools, suchas, for example, RNA interference (RNAi) including non-coding RNAs suchas small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNAs(miRNA), or other nucleotide-based molecules. In an embodiment, siRNAsin the order of 14-27 nucleotides in length are used for genesuppression. ShRNAs can be used to express functional siRNAsintracellularly and to achieve suppression in vitro and in vivo. Othersuppression molecules include, for example, sense and antisense nucleicacids (single or double stranded), ribozymes, peptides, DNAzymes,peptide nucleic acids (PNAs), triple helix forming oligonucleotides,antibodies, and aptamers and modified form(s) thereof directed tosequences in gene(s), RNA transcripts, or proteins.

In an embodiment, the invention relates to vector(s) for supplying anendogenously generated suppression agent, such as, for example, a dsRNAin the form of a short hairpin (shRNA) which can be processedintracellularly into siRNA. dsRNA may be locally or systemicallydelivered. Expression vectors are used to generate functional siRNAs incells and in animals typically using polymerase III promoters to driveexpression, although polymerase II promoters are also used. For example,miRNA structures can be used to express double stranded RNAs frompolymerase II promoters to enable tissue specific expression of doublestranded RNA or polymerase II promoters can be juxtaposed to shRNAsequences to be expressed.

Suppression agents may be modified to alter the potency of thesuppression agent, the target affinity of the suppression agent, thesafety profile of the suppression agent and/or the stability of thesuppression agent, for example, to render them resistant or partiallyresistant to intracellular degradation. Modifications, such asphosphorothioates, for example, can be made to oligonucleotides toincrease resistance to nuclease degradation, binding affinity and/oruptake. In addition, hydrophobization and bioconjugation enhances siRNAdelivery and targeting (De Paula et al., RNA. 13(4):431-56, 2007) andsiRNAs with ribo-difluorotoluyl nucleotides maintain gene silencingactivity (Xia et al., ASC Chem. Biol. 1(3):176-83, (2006). siRNAs withamide-linked oligoribonucleosides have been generated which are moreresistant to 51 nuclease degradation (Iwase R et al. 2006 Nucleic AcidsSymp Ser 50: 175-176). In addition, modification of siRNA at the2′-sugar position and phosphodiester linkage confers improved serumstability without loss of efficacy (Choung et al., Biochem. Biophys.Res. Commun. 342(3):919-26, 2006). In one study,2′-deoxy-2′-fluoro-beta-D-arabinonuclecic acid (FANA)-containingantisense oligonucleotides compared favourably to phosphorothioateoligonucleotides, 2′-0-methyl-RNA/DNA chimeric oligonucleotides andsiRNAs in terms of suppression potency and resistance to degradation(Ferrari N et al. 2006 Ann N Y Acad Sci 1082: 91-102.)

Antisense and ribozyme suppression strategies have led to the reversalof a tumor phenotype by reducing expression of a gene product or bycleaving a mutant transcript at the site of the mutation (Carter andLemoine Br. J. Cancer. 67(5):869-76, 1993; Lange et al., Leukemia.6(11):1786-94, 1993; Valera et al., J. Biol. Chem. 269(46):28543-6,1994; Dosaka-Akita et al., Am. J. Clin. Pathol. 102(5):660-4, 1994; Fenget al., Cancer Res. 55(10):2024-8, 1995; Quattrone et al., Cancer Res.55(1):90-5, 1995; Lewin et al., Nat Med. 4(8):967-71, 1998). Forexample, neoplastic reversion was obtained using a ribozyme targeted toan H-ras mutation in bladder carcinoma cells (Feng et al., Cancer Res.55(10):2024-8, 1995). Ribozymes have also been proposed as a means ofboth inhibiting gene expression of a mutant gene and of correcting themutant by targeted trans-splicing (Sullenger and Cech Nature371(6498):619-22, 1994; Jones et al., Nat. Med. 2(6):643-8, 1996).Ribozyme activity may be augmented by the use of, for example,non-specific nucleic acid binding proteins or facilitatoroligonucleotides (Herschlag et al., Embo J. 13(12):2913-24, 1994;Jankowsky and Schwenzer Nucleic Acids Res. 24(3):423-9, 1996).Multitarget ribozymes (connected or shotgun) have been suggested as ameans of improving efficiency of ribozymes for gene suppression (Ohkawaet al., Nucleic Acids Symp Ser. (29):121-2, 1993).

Triple helix approaches have also been investigated forsequence-specific gene suppression. Triplex forming oligonucleotideshave been found in some cases to bind in a sequence-specific manner(Postel et al., Proc. Natl. Acad. Sci. U.S.A. 88(18):8227-31, 1991;Duval-Valentin et al., Proc. Natl. Acad. Sci. U.S.A. 89(2):504-8, 1992;Hardenbol and Van Dyke Proc. Natl. Acad. Sci. U.S.A. 93(7):2811-6, 1996;Porumb et al., Cancer Res. 56(3):515-22, 1996). Similarly, peptidenucleic acids have been shown to inhibit gene expression (Hanvey et al.,Antisense Res. Dev. 1(4):307-17, 1991; Knudsen and Nielson Nucleic AcidsRes. 24(3):494-500, 1996; Taylor et al., Arch. Surg. 132(11):1177-83,1997). Minor groove binding polyamides can bind in a sequence-specificmanner to DNA targets and hence may represent useful small molecules forfuture suppression at the DNA level (Trauger et al., Chem. Biol.3(5):369-77, 1996). In addition, suppression has been obtained byinterference at the protein level using dominant negative mutantpeptides and antibodies (Herskowitz Nature 329(6136):219-22, 1987;Rimsky et al., Nature 341(6241):453-6, 1989; Wright et al., Proc. Natl.Acad. Sci. U.S.A. 86(9):3199-203, 1989). In some cases suppressionstrategies have lead to a reduction in RNA levels without a concomitantreduction in proteins, whereas in others, reductions in RNA have beenmirrored by reductions in protein.

The diverse array of suppression strategies that can be employedincludes the use of DNA and/or RNA aptamers that can be selected totarget, for example, a protein of interest such as rhodopsin. In thecase of age related macular degeneration (AMD), anti-VEGF aptamers havebeen generated and have been shown to provide clinical benefit in someAMD patients (Ulrich H, et al. Comb. Chem. High Throughput Screen 9:619-632, 2006). Suppression and replacement using aptamers forsuppression in conjunction with a modified replacement gene and encodedprotein that is refractory or partially refractory to aptamer-basedsuppression could be used in the invention.

Recent evidence suggests that control of gene expression occursendogenously in part by the activity of small non-coding RNAs, one broadcategory of which is termed microRNAs (miRNAs). miRNAs are expressedfrom polymerase II promoters, but can also be expressed from polymeraseIII promoters. miRNAs are processed intracellularly from largertranscripts to form small molecules approximately 20 nucleotides inlength. miRNA structures can be used to express small double strandedRNAs and thus can be used to express the double stranded RNAs of thecurrent invention.

Suppression targeted to coding sequence holds the advantage that suchsequences are present in both precursor and mature RNAs, therebyenabling suppressor effectors to target all forms of RNA. A combinedapproach using a number of suppression effectors directed to multipletargets on an RNA or to multiple RNAs may also be used in the invention.As with suppression, multiple replacement nucleic acids can be used inthe invention. For some disorders, it may be necessary to blockexpression of a disease allele completely to prevent disease symptomswhereas for others low levels of mutant protein may be tolerated. Theinvention can thus provide partial or complete suppression.

In one embodiment of the invention, suppressors are targeted to genesthat are involved in the regulation of other genes. Suppression of thesegenes therefore may lead to up- or down-regulation of other genes.

In another embodiment, the invention relates to suppression of theexpression of mutated genes that give rise to a dominant or deleteriouseffect or disease. A suppression effector may target either the diseaseallele or the normal allele. In another embodiment, the suppressioneffector targets both the disease allele and the normal allele.

In an embodiment of the invention, a replacement nucleic acid isprovided that is altered at one or more degenerate or wobble bases fromthe endogenous wild type gene but that encodes the identical amino acidsas the wild type or a non-disease causing gene. In another embodiment,the replacement nucleic acid encodes a beneficial replacement nucleicacid (e.g., a more active or stable product than that encoded by thewild-type gene). The replacement nucleic acid provides expression of anormal or non-disease causing protein product when required toameliorate pathology associated with reduced levels of wild typeprotein. The same replacement nucleic acid can be used in conjunctionwith the suppression of many different disease mutations within a givengene. In addition, multiple replacement nucleic acids can be used in theinvention.

Although the instant application provides numerous exemplary suppressionagents and replacement nucleic acid sequences, these are only examplesand other such sequences can be determined as described herein for thesame targets or for any desired target. “Functional variant” includesany variant nucleic acid or other suppression agent that may have one ormore nucleic acid substitutions but that does not have a materiallydifferent function than, or that can still hybridize under stringenthybridization conditions (0.2×SCC, 0.1% SDS) to, or that shares at least70% identity, for example 80%, such as at least 90% or at least 95%sequence identity with the nucleic acid indicated.

In another embodiment of the invention, suppression effectors aretargeted to the untranslated regions (either 5′UTR or 3′UTR) of at leastone allele of a gene. In another embodiment of the invention replacementnucleic acids are provided that have been altered at the suppressionsite, such that replacement nucleic acids provide functional orpartially functional protein and escape or partially escape fromsuppression by suppressors.

In another embodiment of the invention, suppression effectors aretargeted to intronic sequences. In another embodiment, replacementnucleic acids are provided which have been altered at one or morenucleotides of the targeted site of the intron so that transcripts fromthe replacement nucleic acids escape or partially escape suppression bysuppressors. In another embodiment the whole targeted intron may not bepresent in replacement nucleic acids.

In another embodiment of the invention, suppression effectors aretargeted to polymorphic sites and at least one allele of the gene issuppressed or partially suppressed. In another embodiment, replacementnucleic acids are provided for the alternative polymorphic variant suchthat replacement nucleic acids encode functional or partially functionalprotein and escape or partially escape from suppression by suppressors.

In another embodiment of the invention the suppression agent and/orreplacement nucleic acid is expressed from one or more promotersequences. The invention provides promoter sequences that have beendemonstrated to promote ubiquitous expression of nucleotides and/orpromoters that have been demonstrated to exert tissue specific,temporal, inducible, and/or quantitative control of gene expression. Theinvention also provides enhancer sequences (Table 1) and/orpost-translational regulatory elements and/or other regulatory elementsand/or epigenetic elements that provide optimized expression ofsuppression agents and/or replacement nucleic acids.

TABLE 1 Exemplary Enhancer Elements Enhancer Element Reference Chickenovalbumin upstream promoter Eguchi et al., Biochimie transcriptionfactor II 89(3): 278-88, 2007 Mouse dystrophin muscle promoter/enhancerAnderson et al., Mol. Ther. 14(5): 724-34, 2006 Tobacco eIF4A-10promoter elements Tian et al., J. Plant Physiol. 162(12): 1355-66, 2005Immunoglobulin (Ig) enhancer element Frezza et al., Ann. Rheum. HS1,2ADis. Mar. 28, 2007 Col9a1 enhancer element Genzer and BridgewaterNucleic Acids Res. 35(4): 1178-86, 2007 Gata2 intronic enhancerKhandekar et al., Development Mar. 29, 2007 TH promoter enhancer Gao etal., Brain Res. 1130(1): 1-16, 2007 CMV enhancer InvivoGen cat#pdrive-cag 05A13-SV Woodchuck hepatitis virus Donello et al., J. Virol.posttranscriptional regulatory element 72(6): 5085-92, 1998 Woodchuckhepatitis virus Schambach et al., Gene posttranscriptional regulatoryelement Ther. 13(7): 641-5, 2006 IRBP Ying et al., Curr. Eye Res. 17(8):777-82, 1998 CMV enhancer and chicken β-actin promoter InvivoGen cat#pdrive-cag 05A13-SV CMV enhancer and chicken β-actin promoter InvivoGencat# pdrive-cag and 5′UTR 05A13-SV CpG-island Antoniou et al., Genomics82: 269-279, 2003

In a particular embodiment, sequences that influence chromatinstructure, such as but not exclusive to insulator, antirepressor,cis-acting modulators of nucleosome positioning and/or silencerelements, sometimes termed epigenetic elements, are used to modulateexpression of suppression agents and/or replacement nucleic acids.Exemplary epigenetic elements such as insulator and antirepressorsequences are provided in Table 2. It is clear that chromatin structuresinfluence gene expression, for example, chromatin structures influencethe ability of the transcriptional machinery to access promoter and/orenhancer elements amongst other sequence motifs. The inclusion ofsequences which influence chromatin structures in viral and/or non-viralvectors and/or administered in conjunction with suppression and/orreplacement nucleic acids can be used to optimize expression of eitheror both suppressors and replacement nucleic acids. In addition, chemicalentities which influence chromatin structures can be used to optimizeexpression such as histone deacetylase (HDAC) inhibitors and/or DNAmethyl transferase inhibitors and/or histone methyl transferaseinhibitors. Such entities can be supplied in the form of DNA and/or RNAand/or protein amongst other forms. Similarly attracting enzymes and/orsupplying enzymes (in the form of DNA and/or RNA and or protein)involved in chromatin remodelling such as but not exclusive to histoneacetyl transferases to nucleic acids to be expressed and theirassociated regulatory regions can be used to optimize expression ofsuppression and/or replacement nucleic acids.

TABLE 2 Exemplary Epigenetic Elements Epigenetic elements Reference McpInsulators Kyrchanova et al., Mol. Cell Biol. 27(8): 3035-43, 2007CpG-island region of the HNRPA2B1 Williams et al., BMC Biotechnol. locus5: 17, 2005 Chicken b-globin 5′hypersensitive Kwaks and Otte 2006 Trendsin site 4 (cHS4) Biotechnology 24: 137-142 Ubiquitous chromatin openingKwaks and Otte 2006 Trends in elements (UCOEs) Biotechnology 24: 137-142Matrix associated regions (MARs) Kwaks and Otte 2006 Trends inBiotechnology 24: 137-142 Stabilising and antirepressor Kwaks and Otte2006 Trends in elements (STAR) Biotechnology 24: 137-142 Human growthhormone gene Trujillo MA et al. 2006 Mol silencer Endocrinol 20: 2559S/MAR Liebich et al., Nucleic Acids Res. 30: 3433-42, 2002

In another embodiment, expression of a suppression agent and/orreplacement nucleic acid is optimized to enable efficient suppression inconjunction with sufficient replacement. In an additional embodiment,suppression and/or replacement nucleic acids are provided with agentsthat aid vector transfection, transduction, and/or expression ofsuppression and replacement nucleic acids.

The invention circumvents the need for a specific therapy for everydisease-causing mutation within a given gene. Notably, the invention hasthe advantage that the same suppression agents can be used to suppressmany mutations in a gene. This is particularly relevant when any one ofa large number of mutations within a single gene can cause diseasepathology. The compositions and methods of the invention allow greaterflexibility in choice of target sequence for suppression of expressionof a disease allele. Furthermore, the compositions and methods of theinvention allow greater flexibility in terms of controlling expressionof the suppression and/or replacement of a given gene and or allele of agene.

Suppression and replacement can be undertaken in conjunction with eachother or separately. Suppression and replacement utilizing thedegeneracy of the genetic code may be undertaken in test tubes, incells, in animals, or in plants and may be used for experimentalresearch (e.g., for the study of development or gene expression) or fortherapeutic purposes. Suppression and replacement may be used inconjunction with agents to promote cell transfection or celltransduction such as, for example, lipids and polymers. Suppression andreplacement may be provided to consumers in a kit.

The suppression and replacement agents of the invention can be deliveredto a target cell and or tissue and or animal and or plant using ‘naked’reagents such as DNA, RNA, peptides or other reagents. Alternativelyviral and or non-viral vectors can be used with or without ‘naked’reagents.

In an embodiment, suppression and/or replacement construct(s) can bedelivered to a cell using an AAV2/5 recombinant virus, however, otherviral and non-viral vectors, such as other AAV serotypes, adenovirus,herpes virus, SV40, HIV, SIV and other lentiviral vectors, RSV andnon-viral vectors including naked DNA, plasmid vectors, peptide-guidedgene delivery, terplex gene delivery systems, calcium phosphatenanoparticles, magnetic nanoparticles, colloidal microgels and/or theintegrase system from bacteriophage phiC31 may be utilised in theinvention, for example. Suppression and replacement components may befound on separate vectors or may be incorporated into the same vector.Viral vectors useful in the invention include, but are not limited to,those listed in Table 3. Non-viral vectors useful in the inventioninclude, but are not limited to, those listed in Table 4. Cationiclipid-based non-viral vectors can include glycerol-based (e.g. DOTMA,DOTAP, DMRIE, DOSPA), non-glycerol-based (e.g. DOGS, DOTIM) and/orcholesterol-based cationic lipids (e.g. BGTC, CTAP; Karmali PP andChaudhuri A 2006 Med Res Rev). Viral and non-viral vector delivery maybe accompanied by other molecules such as cationic lipids and/orpolymers and/or detergents and/or agents to alter pH, such as, forexample, polyethelene glycol (PEG), to enhance cellular uptake ofvectors and/or to enhance expression from vectors and/or to evade theimmune system. For example, polycationic molecules have been generatedto facilitate gene delivery including but not exclusive to cationiclipids, poly-amino acids, cationic block co-polymers, cyclodextrinsamongst others. Pegylation of vectors with polyethelene glycol (PEG) canshield vectors from, for example, the extracellular environment. Vectorsmay be used in conjunction with agents to avoid or minimise cellularimmune responses such as PEG or as a Polyplex with Poly(L-Lysine) Vectordelivery may be undertaken using physical methodologies such aselectroporation, nucleofection and/or ionotophoresis, either alone or incombination with molecules to enhance delivery. Vectors may be used inconjunction with agents to promote expression of suppression and/orreplacement components incorporated into vectors, for example, usinghistone deacetylase inhibitors (HDAC) and/or DNA methyl transferaseinhibitors and/or histone methyl transferase inhibitors to modulatechromatin structures thereby aiding expression. HDAC inhibitors includebut are not exclusive to short chain fatty acids such as valproic acidand sodium butyrate, ketones, benzamides, cyclic and non-cyclichydroxamates such as suberoyl anilide hydroxamic acids (SAHA),trichostatin A (TSA), cyclic peptides or tetrapeptides amongst others(Liu T et al. 2006 Cancer Treatment Reviews 32: 157-165). DNA methyltransferase inhibitors including, for example, 5-AC, decitabine andzebularine can be used to modulate chromatin structures. In addition,histone methyl transferase inhibitors can influence chromatin states,for example, BIX-01294 (diazepin-quinazolin-amine derivative). Inaddition, to the chemical entities referred to above, nucleicacids-based inhibitors can be used to suppress expression of proteinsand/or non-coding RNAs involved in chromatin remodelling. In oneembodiment of the invention vectors are optimized to specificallytransduce target cell type(s) or target tissue type(s). Viral and/ornon-viral vectors may be modified to target specific cell types and/orto prevent targeting of some cell types. For example, the inclusion ofthe capsid from AAV serotype 5 in an AAV2/5 hybrid virus facilitatestransduction of photoreceptor cells. Similarly, for example, peptidesmay be included in viral vectors to facilitate targeting. Syntheticnon-viral vectors can be modified to include ligands to facilitatetargeting of vectors to specific cell and/or tissue types, for example,folate can be conjugated to liposomes to target tumour cells which overexpress the folate receptor (Hattori Y et al. 2005; Curr Drug Deliv 3:243-52). In another embodiment of the invention, suppression andreplacement vectors are designed to optimize the generation and/orproduction of vector, for example, to optimize viral titre and/or tooptimize the number or type of nucleotides incorporated into vector(s).For example, vector genomes may be modified such that large transgenesmay be incorporated into vectors, for example, ‘gutless’ adenovirusvectors have an increased capacity in terms of size than previousgenerations of adenovirus vectors. Components of vectors can be modifiedto optimize generation and production of vectors, for example, genesinvolved in replication of AAV can be modified to optimize replicationand/or self complementary AAV vectors can be used to optimize rates oftransgene expression. In an additional embodiment, vectors are designedto optimize suppression in conjunction with replacement, to enableoptimal expression of all components of a therapeutic. For example, tooptimize expression of both elements of suppression and replacement froma given vector, additional sequences can be included in the vector. Forexample, inclusion of nucleotides to separate the ITRs of AAV and theshRNA sequences of an RNAi-based suppression agent can result inoptimisation of expression of the suppression component. Nucleotidesencoding suppressors and/or replacement nucleic acids can be juxtaposedor separated from each other and/or can be in the same orientation oropposing orientations. In addition, the suppressor(s) can be 5′ and/or3′ to the replacement nucleic acids. Nucleotides encoding suppressorsand/or replacement nucleic acids can be juxtaposed to nucleotidescomprising vector(s) or can be separated from nucleotides comprisingvector(s). Nucleotides encoding suppressors and/or replacement nucleicacids may be cloned within the backbone of the plasmid used to generateAAV and or may be cloned between the AAV ITRs and not within the plasmidbackbone of the plasmid, and/or may be cloned in a combination of thesepositions. Additional sequences, such as, for example, stuffer sequencescan be included in vectors to optimize vector design. In addition,multiple suppressors and/or replacement nucleic acids may be used in onevector.

TABLE 3 Exemplary Viral Vectors Delivery Method Serotype Reference AAVAll serotypes, Lebkowski et al., Mol. including but Cell Biol. 8(10):3988- not limited to 96, 1988 1, 2, 3, 4, 5, 6, Flannery et al., Proc.7, 8, 9, 10, 11, Natl. Acad. Sci. U.S.A. 12, 94(13): 6916-21, 1997Lentivirus (for example VSV-G Pang et al., Mol. Vis. but not exclusivelyRabies-G 12: 756-67, 2006 Feline—FIV, Further Takahashi Methods Mol.Equine—EIAV, serotypes** Biol. 246: 439-49, 2004 Bovine—BIV and Balagganet al., J. Gene Simian—SIV). Med. 8(3): 275-85, 2006 Adenovirus VariousBennett et al., Nat. Med. 2(6): 649-54, 1996 Simian papovirius SV40Various Kimchi-Sarfaty et al., Hum. Gene Ther. 13(2): 299-310, 2002Semliki Forest Virus Various DiCiommo et al., Invest. Ophthalmol. Vis.Sco. 45(9): 3320-9, 2004 Sendai Virus Various Ikeda et al., Exp. EyeRes. 75(1): 39-48, 2002

The list provided is not exhaustive; other viral vectors andderivatives, natural or synthesized could be used in the invention.

TABLE 4 Exemplary Non-Viral Vectors or Delivery Methods Delivery MethodReference Cationic Sakurai et al., Gene Ther. 8(9): 677-86, liposomes2001 HVJ liposomes Hangai et al., Arch. Ophthalmol. 116(3): 342-8, 1998Polyethylenimine Liao and Yau Biotechniques 42(3): 285- 6, 2007 DNAnanoparticles Farjo et al., PloS ONE 1: e38, 2006 Dendrimers Marano etal., Gene Ther. 12(21): 1544-50, 2005 Bacterial Brown and Giaccia CancerRes. 58(7): 1408-16, 1998 Macrophages Griffiths et al., Gene Ther. 7(3):255-62, 2000 Stem cells Hall et al., Exp. Hematol. 34(4): 433-42, 2006Retinal transplant Ng et al., Chem. Immunol. Allergy 92: 300-16, 2007Marrow/Mesenchymal Kicic et al., J. Neurosci. 23(21): 7742-9, stromalcells 2003 Chng et al., J. Gene Med. 9(1): 22-32, 2007 Implant (e.g.,Montezuma et al., Invest. Ophthalmol. Vis. Poly(imide)uncoated Sci.47(8): 3514-22, 2006 or coated) Electroporation Featherstone A.Biotechnol. Lab. 11(8): 16, 1993 Targeting peptides Trompeter et al., J.Immunol Methods. (for example but 274(1-2): 245-56, 2003 not exclusivelyTat) Lipid mediated (e.g., Nagahara et al., Nat. Med. 4(12): 1449-52,DOPE, PEG) 1998 Zeng et al., J. Virol. 81(5): 2401-17, 2007 Caplen etal., Gene Ther. 2(9): 603-13, 1995Manconi et al., Int. J. Pharm. 234(1-2): 237-48, 2006 Amrite et al., Invest. Ophthalmol. Vis. Sci. 47(3):1149-60, 2006 Chalberg et al., Invest. Ophthalmol. Vis. Sci. 46(6):2140-6, 2005

The list provided is not exhaustive. Other non-viral vectors andderivatives, natural or synthesized and other delivery methods could beused with the invention.

In an embodiment, the replacement nucleic acid encodes mammalianrhodopsin, collagen 1A1, collagen 1A2, collagen 7A1, or peripherin. Inanother embodiment, the replacement nucleic acid encodes a protein thathas been mutated to cause an autosomal or X-linked dominant retinitispigmentosa, such as those listed in Table 5. Suppression agents andreplacement nucleic acids may be generated for one or more of thesegenes, for example.

TABLE 5 Genes known to be involved in retinitis pigmentosa (Tableadapted from RETNET) (http://www.sph.uth.tmc.edu/Retnet/) Symbols; OMIMNumbers Location Diseases; Protein References LCA9 1p36 recessive Lebercongenital Keen et al., Hum. Mol. amaurosis Genet. 3: 367-368 (1994)NPHP4, 1p36.31 recessive Senior-Loken Mollet et al., Nat. SLSN4;syndrome; recessive Genet. 32: 300-305 nephronophthisis, juvenile;(2002); Otto et al., Am. protein: nephronophthisis 4 J. Hum. Genet.protein 71: 1161-1167 (2002); Schuermann et al., Am. J. Hum. Genet. 70:1240-1246 (2002) RP32; 1p34.3-p13.3 recessive RP, severe Zhang et al.,Hum. Genet. 118: 356-365 (2005) RPE65, 1p31.2 recessive Leber congenitalAcland Nat. Genet. LCA2, RP20; amaurosis; recessive RP; protein: 28:92-95 (2001) retinal pigment epithelium- specific 65 kD protein ABCA4,1p22.1 recessive Stargardt disease, Lewis et al., Am. J. ABCR, RP19,juvenile and late onset; recessive Hum. Genet. 64: 422- STGD1; MD;recessive RP; recessive 1434 (1999) fundus flavimaculatus; recessivecone-rod dystrophy; protein: ATP-binding cassette transporter - retinalCOL11A1, 1p21.1 dominant Stickler syndrome, type Annunen et al., Am. J.STL2; II; dominant Marshall syndrome; Hum. Genet. 65: 974- protein:collagen, type XI, alpha 1 983 (1999) GNAT2, 1p13.3 recessiveachromatopsia; protein: Aligianis et al., J. Med. ACHM4; guaninenucleotide binding Genet. 39: 656-660 protein (G protein) cone-specifc(2002) transducin alpha subunit PRPF3, 1q21.2 dominant RP; protein:human Chakarova et al., Hum. HPRP3, homolog of yeast pre-mRNA Mol.Genet. 11: 87-92 PRP3, RP18; splicing factor 3 (2002) SEMA4A, 1q22dominant RP; dominant cone-rod Abid et al., J. Med. SEMAB; dystrophy;protein: semaphorin Genet. 43: 378-381 4A (2005) CORD8; 1q23.1-q23.3recessive cone-rod dystrophy Ismail et al., J. Hum. Genet. 51: 827-831(2006) AXPC1 1q31-q32 recessive ataxia, posterior column Higgins et al.,Neurol. with RP 52: 146-150 (1999) ARMD1, 1q31.1 dominant MD,age-related; Schultz et al., Hum. FIBL6, protein: hemicentin 1 (fibulin6) Mol. Genet. 12: 3315- FBLN6; 3123 (2003) CFH, HF1; 1q31.3 age-relatedmacular degeneration, Edwards et al., Science complex etiology; protein:308: 421-424 (2005) complement factor H CRB1, RP12; 1q31.3 recessive RPwith para-arteriolar Jacobson et al., Hum. preservation of the RPE(PPRPE); Mol. Genet. 9: 1073- recessive RP; recessive Leber 1078 (2003)congenital amaurosis; dominant pigmented paravenous chorioretinalatrophy; protein: crumbs homolog 1 RD3, 1q32.3 recessive Lebercongenital Friedman et al., Am. J. C1ORF36; amaurosis; protein: RD3protein Hum. Genet. 79: 1059- 1070 (2006) USH2A; 1q41 recessive Ushersyndrome, type Seyedahmadi et al., 2a; recessive RP; protein: usherinExp. Eye. Res. 79: 167- 173 (2004) RP28; 2p16-p11 recessive RP Kumar etal., Mol. Vis. 10: 399-402 (2004) EFEMP1, 2p16.1 dominant radial,macular drusen; Kermani et al., Hum. DHRD, dominant Doyne honeycombGenet. 104: 77-82 MTLV, retinal degeneration (Malattia (1999) FBLN3;Leventinese); protein: EGF- containing fibrillin-like extracellularmatrix protein 1 (fibulin 3) ALMS1, 2p13.1 recessive Alström syndrome;Hearn et al., Nat. ALSS protein: ALMS1 protein Genet. 31: 79-83 (2002)RP33 2cen-q12.1 dominant RP Zhao et al., Hum. Genet. 119: 617-623 (2006)LOC619531 2q11 recessive cone-rod dystrophy and Michaelides et al., J.amelogenesis imperfecta Med. Genet. 41: 468- 473 (2004) CNGA3, 2q11.2recessive achromatopsia; protein: Nishiguchi et al., Hum. ACHM2, conephotoreceptor cGMP-gated Mutat. 25: 248-258 CNCG3, cation channel alphasubunit (2005) RMCH2 MERTK 2q13 recessive RP; protein: c-mer Vollrath etal., Proc. protooncogene receptor tyrosine Natl. Acad. Sci. USA kinase98: 12584-12589 (2001) NPHP1, 2q13 recessive Senior-Loken Hildebrandt etal., Nat. JBTS4, syndrome; recessive Genet. 17: 149-153 SLSN1nephronophthisis, juvenile; (1997) recessive Joubert syndrome; protein:nephronophthisis 3 protein BBS5 2q31.1 recessive Bardet-Biedl syndrome;Li et al., Cell. 117: 541- protein: flagellar apparatus-basal 552 (2004)body protein DKFZp7621194 CERKL, 2q31.3 recessive RP; protein: ceramideTuson et al., Am. J. RP26 kinase-like protein Hum. Genet. 74: 128- 138(2004) SVD 2q36 dominant vitreoretinal Jiao et al., Invest.degeneration, snowflake Ophthalmol. Vis. Sci. 45: 4498-503 (2004) SAG2q37.1 recessive Oguchi disease; Nakazawa et al., Arch. recessive RP;protein: arrestin (s- Ophthalmol. 116: 498- antigen) 501 (1998) USH2B,3p24.2-p23 recessive Usher syndrome, type 2; Hmani et al., Eur. J. DFNB6recessive sensorineural deafness Hum. Genet. 7: 363-367 without RP(1999) CRV, 3p21.3-p21.1 dominant hereditary vascular Ophoff et al., Am.J. HERNS, HVR retinopathy with Raynaud Hum. Genet. 69: 447- phenomenonand migraine 453 (2001) GNAT1 3p21.31 dominant CSNB, Nougaret type;Dryja et al., Nat. Genet. protein: rod transducin alpha 13: 358-360(1996) subunit ATXN7, 3p14.1 dominant spinocerebellar ataxia Aleman etal., Exp. ADCA2, w/MD or retinal degeneration; Eye. Res. 74: 737-745OPCA3, protein: ataxin 7 (2002) SCA7 ARL6, BBS3 3q11.2 recessiveBardet-Biedl syndrome; Fan et al., Nat. Genet. protein: ADP-ribosylationfactor- 36: 989-993 (2004) like 6 IQCB1, 3q13.33 recessive Senior-LokenOtto et al., Nat. Genet. NPHP5, syndrome; protein: IQ motif 37: 282-288(2005) SLSN5 containing B1 protein NPHP3, 3q22.1 recessive Senior-LokenOlbrich et al., Nat. SLSN3 syndrome; recessive Genet. 34: 455-459nephronophthisis, adolescent; (2003) protein: nephronophthisis 3 proteinRHO, RP4 3q22.1 dominant RP; dominant CSNB; Dryja et al., Nat. Genet.recessive RP; protein: rhodopsin 4: 280-283 (1993) RP5 same as RHO notdistinct from RHO/RP4 Farrar et al., Hum. Mol. Genet. 1: 769-771 (1992)USH3A, 3q25.1 recessive Usher syndrome, type 3; Joensuu et al., Am. J.USH3 protein: clarin-1 Hum. Genet. 69: 673- 684 (2001) OPA1 3q29dominant optic atrophy, Kjer Aung et al., Hum. type; dominant opticatrophy with Genet. 110: 52-56 sensorineural hearing loss; (2002)protein: OPA1 protein STGD4 4p dominant Stargardt-like macular Kniazevaet al., Am. J. dystrophy Hum. Genet. 64: 1394- 1399 (1999) MCDR24p16.3-p15.2 dominant MD, bull's-eye Michaelides et al., Invest.Ophthalmol. Vis. Sci. 44: 1657-1662 (2003) PDE6B, 4p16.3 recessive RP;dominant CSNB; Pearce-Kelling et al., CSNB3 protein: rod cGMP Mol. Vis.7: 42-47 phosphodiesterase beta subunit (2001) WFS1, 4p16.1 recessiveWolfram syndrome; Hum. Mol. Genet. DFNA38 dominant low frequency 10:2501-2508 (2001) sensorineural hearing loss; protein: wolframin PROML14p15.32 recessive retinal degeneration; Maw et al., Hum. Mol. protein:prominin (mouse)-like 1 Genet. 9: 27-34 (2000) CNGA1, 4p12 recessive RP;protein: rod cGMP- Dryja et al., Proc. Natl. CNCG, gated channel alphasubunit Acad. Sci. USA CNCG1 192: 10177-10181 (1995) WFS2 4q22-q24recessive Wolfram syndrome; El-Shanti et al., Am. J. dominant Hum.Genet. 66: 1229- 1236 (2000) MTP, ABL 4q23 recessiveabetalipoproteinemia; Narcisi et al., Am. J. protein: microsomaltriglyceride Hum. Genet. 57: 1298- transfer protein 1310 (1995) BBS7,4q27 recessive Bardet Biedl syndrome; Badano et al., Am. J. BBS2L1protein: BBS7 protein Hum. Genet. 72: 650- 658 (2003) BBS12, 4q27recessive Bardet-Biedl syndrome; Stoetzel et al., Am. J. FLJ35630protein: BBS12 protein Hum. Genet. 80: 1-11 (2007) RP29 4q32-q34recessive RP Hameed et al., Invest. Ophthalmol. Vis. Sci. 42: 1436-1438(2001) LRAT 4q32.1 recessive RP, severe early-onset; Thompson et al.,Nat. recessive Leber congenital Genet. 128: 123-124 amaurosis; protein:lecithin (2001) retinol acyltransferase CYP4V2, 4q35.2 recessive Bietticrystalline Li et al., Am. J. Hum. BCD corneoretinal dystrophy; protein:Genet. 74: 817-826 cytochrome P450 4V2 (2004) MCDR3 5p15.33-p13.1dominant MD Michaelides et al., Invest. Ophthalmol. Vis. Sci. 44:2178-2183 (2003) CSPG2, 5q14.3 dominant Wagner disease andKloeckener-Gruissem WGN1, erosive vitreoretinopathy; protein: et al.,Mol. Vis. 12: 350- ERVR chondroitin sulfate proteoglycan 2 355 (2006)(versican) MASS1, 5q14.3 recessive Usher syndrome, type 2; Weston etal., Am. J. USH2C, dominant/recessive febrile Hum. Genet. 74: 357- VLGR1convulsions; protein: monogenic 366 (2004) audiogenic seizuresusceptibility 1 homolog BSMD 5q21.2-q33.2 dominant MD, butterfly-shapedden Hollander et al., J. Med. Genet. 41: 699- 702 (2004) PDE6A 5q33.1recessive RP; protein: cGMP Dryja et al., Invest. phosphodiesterasealpha subunit Ophthalmol. Vis. Sci. 40: 1859-1865 (1999). GRM6 5q35.3recessive CSNB; protein: Dryja et al., Proc. Natl. metabotropicglutamate receptor 6 Acad. Sci. USA 102: 4884-4889 (2005) C2 6p21.32age-related macular degeneration, Gold et al., Nat. Genet. complexetiology; protein: 38: 458-462(2006) complement component 2 CFB, BF,6p21.32 age-related macular degeneration, Gold et al., Nat. Genet. BFDcomplex etiology; protein: 38: 458-462 (2006) complement factor B,properdin TULP1, RP14 6p21.31 recessive RP; recessive Leber Banerjee etal., Nat. congenital amaurosis; protein: Genet. 18: 177-179 tubby-likeprotein 1 (1998) RDS, RP7 6p21.2 dominant RP; dominant MD; Hum. Mutat.10: 301- digenic RP with ROM1; 309 (1997) dominant adult vitelliform MD;protein: peripherin 2 GUCA1A, 6p21.1 dominant cone dystrophy; Payne etal., Am. J. COD3, dominant cone-rod dystrophy; Hum. Genet. 61: A290GCAP1 protein: guanylate cyclase (1997) activating protein 1A GUCA1B,6p21.1 dominant RP; dominant MD; Sato et al., Graefes GCAP2 protein:guanylate cyclase Arch. Clin. Exp. activating protein 1B Ophthalmol.243: 235- 242 (2004) BCMAD 6p12.3-q16 dominant MD, benign concentric vanLith-Verhoeven et annular al., Invest. Ophthalmol. Vis. Sci. 45: 30-35(2004) RP25 6cen-q15 recessive RP Abd El-Aziz et al., Ann. Hum. Genet.(2006) LCA5 6q11-q16 recessive Leber congenital Dharmaraj et al., Am. J.amaurosis Hum. Genet. 66: 319- 326 (2000) COL9A1 6q13 recessive Sticklersyndrome; Van Camp et al., Am. dominant multiple epiphyseal J. Hum.Genet. 79: 449- dysplasia (MED); protein: 457 (2006) collagen, type IX,alpha-1 RIMS1, 6q13 dominant cone-rod dystrophy; Kelsell et al., Am. J.CORD7, protein: regulating synaptic Hum. Genet. 63: 274- RIM1 membraneexocytosis protein 1or 279 (1998) rab3A-interacting molecule MCDR1,6q14-q16.2 dominant MD, North Carolina Small et al., Mol. Vis. PBCRAtype; dominant progressive 5: 38 (1999) bifocal chorioretinal atrophyELOVL4, 6q14.1 dominant MD, Stargardt-like; Edwards et al., Invest.STGD3 protein: elongation of very long Ophthalmol. Vis. Sci. fatty acidsprotein 42: 2652-2663 (2001) AHI1, JBTS3 6q23.3 recessive Joubertsyndrome; Parisi et al., J. Med. protein: Abelson helper Genet. 43:334-339 integration site 1 (2006) PEX7, 6q23.3 recessive Refsum disease,adult van den Brink 0et al., PTS2R, form; protein: peroxisome Am. J.Hum. Genet. RCDP1, biogenesis factor 7 72: 471-477 (2003) RCD1 6q25-q26dominant retinal-cone dystrophy OMIM 07 1 MDDC, 7p21-p15 dominant MD,cystoid Inglehearn et al., Am. J. CYMD Hum. Genet. 55: 581- 582 (1994)PTHB1, 7p14.3 recessive Bardet Biedl syndrome; Nishimura et al., Am. J.BBS9, PHTB1 protein: parathyroid hormone- Hum. Genet. 77: 1021-responsive B1 protein 1033 (2005) RP9, PAP1, 7p14.3 dominant RP;protein: RP9 Sullivan et al., Invest. PIM1K protein or PIM1-kinaseassociated Ophthalmol. Vis. Sci. protein 1 47: 3052-3064 (2006) PEX1,IRD 7q21.2 recessive Refsum disease, Portsteffen et al., Nat. infantileform; protein: Genet. 17: 449-452 peroxisome biogenesis factor 1 (1997)IMPDH1, 7q32.1 dominant RP; dominant Leber Mortimer et al., RP10congenital amaurosis; protein: Biochem. J. 390: 41-47 inosinemonophosphate (2005) dehydrogenase 1 OPN1SW, 7q32.1 dominant tritanopia;protein: blue Fitzgibbon et al., Hum. BCP, CBT cone opsin Genet. 93:79-80 (1994) CORD9 8p11 recessive cone-rod dystrophy Danciger et al.,Invest. Ophthalmol. Vis. Sci. 42: 2458-2465 (2001) RP1 8q12.1 dominantRP; recessive RP; Bowne et al., Hum. protein: RP1 protein Mol. Genet.11: 2121- 2128 (1999) TTPA 8q12.3 recessive RP and/or recessive orYokota et al., New dominant ataxia; protein: alpha- Eng. J. Med. 335:1770- tocopherol-transfer protein 1771 (1996) ROA1 8q21-q22 recessiveoptic atrophy Barbet et al., Eur. J. Hum. Genet. 11: 966- 971 (2003)PXMP3, 8q21.13 recessive Refsum disease, Gartner et al., Nat. PAF1,PEX2, infantile form; protein: Genet. 1: 16-23 (1992) PMP35 peroxisomalmembrane protein 3 CNGB3, 8q21.3 recessive achromatopsia Kohl et al.,Eur. J. ACHM3 Pingelapese; recessive, Hum. Genet. 13: 302- progressivecone dystrophy; 308 (2005) protein: cone cyclic nucleotide- gated cationchannel beta 3 subunit VMD1 not 8q24 dominant MD, atypical Sohocki etal., Am. J. vitelliform Hum. Genet. 61: 239- 241 (1997) RP31 9p22-p13dominant RP Papaioannou et al., Hum. Mut. 118: 501- 503 (2005) KCNV29q24.2 recessive cone dystrophy with Wu et al., Am. J. Hum. supernormalrod Genet. 79: 574-579 electroretinogram; protein: (2006) potasiumchannel subfamily V member 2 INVS, NPHP2 9q31.1 recessive Senior-LokenO'Toole et al., Nephrol. syndrome; recessive Dial. Transplant.nephronophthisis; protein: 21: 1989-1991 (2006) inverson DFNB31 9q32recessive Usher syndrome, type 2; Ebermann et al., Hum. recessivedeafness without RP; Genet. (2006) protein: whirlin TLR4 9q33.1age-related macular degeneration, Zareparsi et al., Hum. complexetiology; protein: toll- Mol. Genet. 14: 1449- like receptor 4 1455(2005) TRIM32, 9q33.1 recessive Bardet-Biedl syndrome; Chiang et al.,Proc. BBS11, HT2A recessive limb-girdle muscular Natl. Acad. Sci. USAdystrophy; protein: tripartite 103: 6287-6292 (2006) motif-containingprotein 32 RP21, RP8 not 9q34-qter dominant RP with sensorineuralMansergh et al., Am. J. deafness Hum. Genet. 64: 971- 985 (1999) JBTS1,9q34 recessive Joubert syndrome Saar et al., Am. J. Hum. CORS1 Genet.65: 1666-1671 (1999) PHYH, 10p13 recessive Refsum disease, adult Jansenet al., Nat. PAHX, RDPA form; protein: phytanoyl-CoA Genet. 17: 190-193hydroxylase (1997) RNANC 10q21 recessive nonsyndromal Ghiasvand et al.,Am. J. congenital retinal nonattachmen Med. Genet. 90: 165- 168 (2000)PCDH15, 10q21.1 recessive Usher syndrome, type Ahmed et al., Hum.DFNB23, 1f; recessive deafness without Mol. Genet. 12: 3215- USH1F RP;protein: protocadherin 15 3223 (2003) CDH23, 10q22.1 recessive Ushersyndrome, type Astuto et al., Am. J. DFNB12, 1d; recessive deafnesswithout Hum. Genet. 71: 262- USH1D RP; protein: cadherin-like gene 275(2002) 23 RGR 10q23.1 recessive RP; dominant choroidal Morimura et al.,Nat. sclerosis; protein: RPE-retinal G Genet. 23: 393-394protein-coupled receptor (1999) RBP4 10q23.33 recessive RPEdegeneration; Seeliger et al., Invest. protein: retinol-binding protein4 Ophthalmol. Vis. Sci. 40: 3-11 (1999) PAX2, ONCR 10q24.31 dominantrenal-coloboma Favor et al., Proc. Natl. syndrome; protein: paired Acad.Sci. USA homeotic gene 2 protein 93: 13870-13875 (1996) HTRA1, 10q26.13age-related macular degeneration, DeWan et al., Science PRSS11 complexetiology; protein: HtrA 314: 989-992 (2006) serine peptidase 1 LOC38771510q26.13 age-related macular degeneration, Jakobsdottir et al., Am.complex etiology; protein: J. Hum. Genet. 77: 389- hypothetical proteinwith Entrez 407 (2005) ID 387715 OAT 10q26.13 recessive gyrate atrophy;protein: D Valle, O Simell. In ornithine aminotransferase ‘The Metabolicand Molecular Bases of Inherited Disease’, 8th Ed. C R Schriver, et al.eds., McGraw-Hill. (2000) TEAD1, AA, 11p15.3 dominant atrophia areata;protein: Fossdal et al., Hum. TCF13, TEF1 TEA domain family member 1Mol. Genet. 13: 975- [Entrez] 981 (2004) USH1C, 11p15.1 recessive Ushersyndrome, Ahmed et al., Hum. DFNB18 Acadian; recessive deafness Genet.110: 527-531 without RP; protein: harmonin (2002) EVR3 11p13-p12dominant familial exudative Downey et al., Am. J. vitreoretinopathy Hum.Genet. 68: 778- 781 (2001) CORS2, 11p12-q13.3 recessive Joubert syndromeValente et al., Ann. JBTS2 Neurol. 57: 513-519 (2005) ROM1 11q12.3dominant RP; digenic RP with Dryja et al., Invest. RDS; protein: retinalouter Ophthalmol. Vis. Sci. segment membrane protein 1 18: 1972-1982(1997) VMD2 11q12.3 dominant MD, Best type; Weber et al., Am. J.dominant Hum. Genet. 55: 1182- vitreoretinochoroidopathy; 1187 (1994a)protein: bestrophin BBS1 11q13 recessive Bardet-Biedl syndrome; Mykytynet al., Nat. protein: BBS1 protein Genet. 31: 435-438 (2002) VRNI 11q13dominant neovascular Stone et al., Hum. Mol. inflammatoryvitreoretinopathy Genet. 1: 685-689 (1992) CABP4 11q13.1 recessive CSNB;protein: calcium Zeitz et al., Am. J. binding protein 4 Hum. Genet. 79:657- 667 (2006) LRP5, EVR4, 11q13.2 dominant familial exudative Jiao etal., Am. J. Hum. HBM, OPPG vitreoretinopathy; dominant high Genet. 75:878-884 bone mass trait; recessive (2004) osteoporosis-pseudogliomasyndrome; recessive FEVR; protein: low density lipoproteinreceptor-related protein 5 MYO7A, 11q13.5 recessive Usher syndrome, type1 ; Gibbs et al., Natl. DFNB2, recessive congenital deafness Acad. Sci.USA USH1B without RP; recessive atypical 100: 6481-6486 (2003) Ushersyndrome (USH3-like); protein: myosin VIIA FZD4, EVR1, 11q14.2 dominantfamilial exudative Müller et al., Genomics FEVR vitreoretinopathy;protein: 20: 317-319(1994) frizzled-4 Wnt receptor homolog C1QTNF5,11q23.3 dominant MD, late onset; Ayyagari et al., Invest. CTRP5 dominantMD with lens zonules; Ophthalmol. Vis. Sci. protein: C1q and tumornecrosis- 46: 3363-3371 (2005) related protein 5 collagen COL2A1,12q13.11 dominant Stickler syndrome, type Snead et al., J. Med. AOM,STL1 I; dominant Wagner syndrome; Genet. 36: 353-659 dominant epiphysealdysplasia; (1999) protein: collagen, type II, alpha 1 RDH5, RDH1 12q13.2recessive fundus albipunctatus; Cideciyan et al., Vis. recessive conedystrophy, late Neurosci. 17: 667-678 onset; protein: 11-cis retinol(2000) dehydrogenase 5 BBS10, 12q21.2 recessive Bardet-Biedl syndrome;Stoetzel et al., Nat. FLJ23560 protein: BBS10 (C12orf58) Genet. 38:521-524 chaperonin (2006) CEP290, 12q21.32 recessive Senior-Loken Changet al., Hum. JBTS5, syndrome; recessive Joubert Mol. Genet. 15: 1847-NPHP6, syndrome; recessive Leber 1857 (2006) SLSN6 congenital amaurosis;protein: centrosomal protein 290 kDa RB1 13q14.2 dominant germline orsomatic Lohmann et al., Am. J. retinoblastoma; benign retinoma; Hum.Genet. 58: 940- pinealoma; osteogenic sarcoma; 949 (1996) protein:retinoblastoma protein 1 GRK1, 13q34 recessive CSNB, Oguchi type;Cideciyan et al., Proc. RHOK, RK protein: rhodopsin kinase Natl. Acad.Sci. USA 95: 328-333 (1998) STGD2 not 13q34 dominant MD, Stargardt typeZhang et al., Nat. Genet. 27: 89-93 (2001) ACHM1, 14 recessive rodmonochromacy or Pentao et al., Am. J. RMCH achromatopsia Hum. Genet. 50:690- 699 (1992) RP16 not 14 recessive RP Bruford et al., Am. J. Hum.Genet. 55: A181 (1994) MCDR4 14q dominant MD, North Carolina- Francis etal., Br. J. like with progressive Ophthalmol. 87: 893- sensorineuralhearing loss 898 (2003) NRL, RP27 14q11.2 dominant RP; recessive RP;Mears et al., Nat. protein: neural retina lucine Genet. 29: 447-452zipper (2001) RPGRIP1, 14q11.2 recessive Leber congenital Mellersh etal., LCA6 amaurosis; protein: RPGR- Genomics 88: 293-301 interactingprotein 1 (2006) LCA3 14q24 recessive Leber congenital Stockton et al.,Hum. amaurosis Genet. 103: 328-333 (1998) RDH12 14q24.1 recessive Lebercongenital Janecke et al., Nat. amaurosis with severe childhood Genet.36: 850-854 retinal dystrophy; protein: retinol (2004) dehydrogenase 12USH1A, not 14q32 recessive Usher syndrome, Gerber et al., Am. J. USH1French Hum. Genet. 78: 357- 359 (2006) TTC8, BBS8 14q32.11 recessiveBardet-Biedl syndrome; Ansley et al., Nat. protein: tetratricopeptiderepeat 425: 628-633 (2003) domain 8 FBLN5 14q32.12 familial MD,age-related; protein: Arch. Ophthalmol. fibulin 5 112: 765-772 (1994)NR2E3, 15q23 recessive enhanced S-cone Sharon et al., Arch. ESCS, PNRsyndrome; recessive RP in Ophthalmol. 121: 1316- Portuguese Crypto Jews;1323 (2003) Goldmann-Favre syndrome; protein: nuclear receptor subfamily2 group E3 MRST 15q24 recessive retardation, spasticity Mitchell et al.,Am. J. and retinal degeneration Hum. Genet. 62: 1070- 1076 (1998) BBS415q24.1 recessive Bardet-Biedl syndrome; Katsanis et al., Nat. protein:BBS4 protein Genet. 26: 67-70 (2000) RLBP1, 15q26.1 recessive RP;recessive Bothnia Burstedt et al., Invest. CRALBP dystrophy; recessiveretinitis Ophthalmol. Vis. Sci. punctata albescens; recessive 40:995-1000 (1999) Newfoundland rod-cone dystrophy; protein: retinaldehyde-binding protein 1 ABCC6, 16p13.11 recessive pseudoxanthoma Bergen etal., Nat. ARA, MRP6, elasticum; dominant Genet. 25: 228-231 PXEpseudoxanthoma elasticum; (2000) protein: ATP-binding casette, subfamilyC, member 6 RP22 16p12.3-p12.1 recessive RP Finckh et al., Genomics 48:341-345 (1998) CLN3, JNCL 16p11.2 recessive Batten disease (ceroid-Kremmidiotis et al., lipofuscinosis, neuronal 3), Hum. Mol. Genet.juvenile; protein: Batten disease 8: 523-531 (1999) protein BBS2 16q12.2recessive Bardet-Biedl syndrome; Beales et al., Am. J. protein: BBS2protein Hum. Genet. 68: 606- 616 (2001) CNGB1, 16q13 recessive RP;protein: rod cGMP- Bareil et al., Hum. CNCG2, gated channel beta subunitGenet. 108: 328-334 CNCG3L, (2001) GAR1, GARP CDH3, 16q22.1 recessiveMD, juvenile with Indelman et al., J. CDHP, PCAD hypotrichosis; protein:cadherin 3, Invest. Dermatol. type 1, placental 119: 1210-1213 (2002)FHASD 16q23.2-q24.2 recessive foveal hypoplasia and Pal et al., J. Med.anterior segment dysgenesis Genet. 41: 772-777 (2004) CACD 17p13dominant central areolar Hughes et al., J. Med. choroidal dystrophyGenet. 35: 770-772 (1998) PRPF8, 17p13.3 dominant RP; protein: humanKojis et al., Am. J. PRPC8, RP13 homolog of yeast pre-mRNA Hum. Genet.58: 347- splicing factor C8 355 (1996) AIPL1, LCA4 17p13.2 recessiveLeber congenital Hanein et al., Hum. amaurosis; dominant cone-rod Mutat.23: 306-317 dystrophy; protein: (2004) arylhydrocarbon-interactingreceptor protein-like 1 GUCY2D, 17p13.1 recessive Leber congenitalHanein et al., Hum. CORD6, amaurosis; dominant cone-rod Mutat. 23:306-317 LCA1, dystrophy; protein: retinal- (2004) RETGC, specificguanylate cyclase RETGC1 CORD5, same as dominant cone-rod dystrophy,Udar et al., Hum. Mut. RCD2 GUCY2D progressive; recessive cone-rod 21:170-171 (2003) dystrophy CORD4 17q cone-rod dystrophy Klystra et al.,UNC119, 17q11.2 dominant cone-rod dystrophy; Kobayashi et al., HRG4protein: human homolog of C. Invest. Ophthalmol. elegans unc119 proteinVis. Sci. 41: 3268-3277 (2000) CA4, RP17 17q23.2 dominant RP; protein:carbonic Rebello et al., Proc. anhydrase IV Natl. Acad. Sci. USA 101:6617-6622 (2004) USH1G, 17q24-q25 recessive Usher syndrome; Kikkawa etal., Hum. SANS protein: human homolog of Mol. Genet. 12: 453- mousescaffold protein containing 461 (2003) ankyrin repeats and SAM domainRGS9 17q24.1 recessive delayed cone Nishiguchi et al., adaptation;protein: regulator of Nature 427: 75-78 G-protein signalling 9 (2004)PRCD 17q25.1 recessive RP; protein: progressive Zangerl et al., rod-conedegneration protein Genomics (2006) FSCN2, RP30 17q25.3 dominant RP;dominant MD; Wada et al., Arch. protein: retinal fascin homolog 2,Ophthalmol. 121: 1613- actin bundling protein 1620 (2003) OPA418q12.2-q12.3 dominant optic atrophy, Kjer type Kerrison et al., Arch.Ophthalmol. 117: 805- 810 (1999) CORD1 18q21.1-q21.3 cone-rod dystrophy;de Grouchy Manhant et al., Am. J. syndrome Hum. Genet. 57: A96 (1995)R9AP 19q13.12 recessive delayed cone Nishiguchi et al., adaptation;protein: regulator of Nature 427: 75-78 G-protein signalling 9-binding(2004) protein MCDR5 19q13.31-q13.32 dominant macular dystrophy Yang etal., Science 314: 992-993 (2006) CRX, CORD2 19q13.32 dominant cone-roddystrophy; Hanein et al., Hum. recessive, dominant and de novo Mutat.23: 306-317 Leber congenital amaurosis; (2004) dominant RP; protein:cone-rod otx-like photoreceptor homeobox transcription factor OPA3, MGA319q13.32 recessive optic atrophy with Anikster et al., Am. J. ataxia and3-methylglutaconic Hum. Genet. 69: 1218- aciduria; protein: OPA3 protein1224 (2001) PRPF31, 19q13.42 dominant RP; protein: human Sullivan etal., Invest. PRP31, RP11 homolog of yeast pre-mRNA Ophthalmol. Vis. Sci.splicing factor 31 47: 4579-4588 (2006) JAG1, AGS 20p12.2 dominantAlagille syndrome; Li et al., Nat. Genet. protein: Jagged protein 1 16:243-251 (1997) MKKS, BBS6 20p12.2 recessive Bardet-Biedl syndrome;Beales et al., Am. J. protein: McKusick-Kaufman Hum. Genet. 68: 606-syndrome protein 616 (2001) PANK2, 20p13 recessive HARP Hartig et al.,Ann. HARP, PKAN (hypoprebetalipoproteinemia, Neurol. 59: 248-256acanthocytosis, RP, and palladial (2006) degeneration); recessiveHallervorden-Spatz syndrome; protein: pantothenate kinase 2 USH1E 21q21recessive Usher syndrome, type 1 Chäib et al., Hum. Mol. Genet. 6: 27-31(1997) OPA5 22q12.1-q12.3 dominant optic atrophy Rozet et al., Invest.Ophthalmol. Vis. Sci. 46: E-Abstract 2292 (2005) TIMP3, SFD 22q12.3dominant Sorsby's fundus Felbor et al., Am. J. dystrophy; protein:tissue Hum. Genet. 60: 57-62 inhibitor of metalloproteinases-3 (1997)RP23 Xp22 X-linked RP Hardcastle et al., Invest. Ophthalmol. Vis. Sci.41: 2080-2086 (2000) RS1, XLRS1 Xp22.13 retinoschisis; protein: Graysonet al., Hum. retinoschisin Mol. Genet. 9: 1873- 1879 (2000) (- - -)Xp21-q21 RP with mental retardation Aldred et al., Am. J. Hum. Genet.55: 916- 922 (1994) RP6 Xp21.3-p21.2 X-linked RP Breuer et al., Invest.Ophthalmol. Vis. Sci. 41: S191 (2000) DMD Xp21.2-p21.1 Oregon eyedisease (probably); D'Souza et al., Hum. protein: dystrophin Mol. Genet.5: 837-842 (1995) AIED, OA2 Xp11.4-q21 Åland island eye disease Wutz etal., Eur. J. Hum. Genet. 10: 449- 456 (2002) COD4 Xp11.4-q13.1 X-linkedprogressive cone-rod Jalkanen et al., J. Med. dystrophy Genet. 40:418-423 (2003) OPA2 Xp11.4-p11.2 X-linked optic atrophy Assink et al.,Am. J. Hum. Genet. 61: 934- 939 (1997) NYX, CSNB1 Xp11.4 X-linked CSNB;protein: Bech-Hansen et al., nyctalopin Nat. Genet. 26: 319-323 (2000)CSNB4 same as NYX X-linked CSNB Pusch et al., Nat. Genet. 26: 324-327(2000) RPGR, RP3 Xp11.4 X-linked RP, recessive; X-linked Bader et al.,Invest. RP, dominant; X-linked CSNB; Ophthalmol. Vis. Sci. X-linked conedystrophy 1; X- 44: 1458-1463(2003) linked atrophic MD, recessive;protein: retinitis pigmentosa GTPase regulator COD1 same as RPGRX-linked cone dystrophy 1 Demirci et al., Am. J. Hum. Genet. 70: 1049-1053 (2002) RP15 same as RPGR X-linked RP, dominant Mears et al., Am. J.Hum. Genet. 67: 1000- 1003 (2000) PRD Xp11.3-p11.23 retinal dysplasia,primary Ravia et al., Hum. Mol. Genet. 8: 1295-1297 (1993) NDP, EVR2Xp11.3 Norrie disease; familial exudative Black et al., Hum. Mol.vitreoretinopathy; Coats disease; Genet. 11: 2021-2035 protein: Norriedisease protein (1999) CACNA1F, Xp11.23 X-linked CSNB, incomplete;Nakamura et al., Arch. CSNB2, ÅIED-like disease; severe CSNB;Ophthalmol. 121: 1028- CSNBX2 protein: L-type voltage-gated 1033 (2003)calcium channel alpha-1 subunit RP2 Xp11.23 X-linked RP; protein: novelHardcastle et al., Am. J. XRP2 protein similar to human Hum. Genet. 64:1210- cofactor C 1215 (1999) PGK1 Xq21.1 RP with myopathy; protein:Tonin et al., Neurol. phosphoglycerate kinase 43: 387-391 (1993) CHMXq21.2 choroideremia; protein: van den Hurk et al., geranylgeranyltransferase Rab Hum. Mutat. 9: 110-117 escort protein 1 (1997) TIMM8A,Xq22.1 optic atrophy with deafness- Koehler et al., Proc. DDP, DDP2,dystonia syndrome; protein: inner Natl. Acad. Sci USA DFN1 mitochondrialmembrane 96: 2141-1246 (1999) translocase 8 homolog A RP24 Xq26-q27X-linked RP Gieser et al., Am. J. Hum. Genet. 63: 1439- 1447 (1998)COD2, Xq27 X-linked progressive cone Bergen et al., XLPCD dystrophy, 2RP34 Xq28-qter X-linked RP Melamud et al., J. Med. Genet. 43: e27 (2006)OPN1LW, Xq28 deuteranopia and rare macular Ayyagari et al., Mol. GCP,CBD dystrophy in blue cone Vis. 58: 98-101 (1999) monochromacy with lossof locus control element; protein: green cone opsin OPN1MW, Xq28protanopia and rare macular Ayyagari et al., Mol. RCP, CBP dystrophy inblue cone Vis. 58: 98-101 (1999) monochromacy with loss of locus controlelement; protein: red cone opsin KSS mitochondrion Kearns-Sayre syndromeincluding al., Science 283: 1482- retinal pigmentary degeneration; 1488(1999) protein: several mitochondrial proteins LHON, mitochondrion Leberhereditary optic Brown et al., Am. J. MTND1, neuropathy; protein:complex I, Hum. Genet. 60: 381- MTND4, III or IV proteins 387 (1997)MTND6 MTTL1, mitochondrion macular pattern dystrophy with Bonte et al.,Retina DMDF, type II diabetes and deafness; 17: 216-221 (1997) TRNL1protein: leucine tRNA 1 (UUA/G), nt 3230-3304 MTATP6, mitochondrion RPwith developmental and White et al., J. Inherit. ATP6, NARP neurologicalabnormalities; Leigh Metab. Dis. 22: 899-914 syndrome; LHON; protein:(1999) complex V ATPase 6 subunit, nt 8527-9207 MTTH, mitochondrionpigmentary retinopathy and Crimi et al., Neurology TRNH sensorineuralhearing loss; 60: 1200-1203 (2003) protein: histidine tRNA, nt12138-12206 MTTS2, mitochondrion RP with progressive sensorineuralMansergh et al., Am. J. TRNS2 hearing loss; protein: serine tRNA Hum.Genet. 64: 971- 2 (AGU/C), nt 12207-12265 985 (1999)

In an embodiment of the invention, suppression agents are siRNAs orshRNAs targeting human rhodopsin. Exemplary siRNAs and replacementrhodopsin sequences are provided in Table 6A.

TABLE 6A Exemplary siRNA Sequences Targeting Human Rhodopsin andReplacement Rhodopsin Sequences SEQ SEQ siRNA Target Site ID NOReplacement Site ID NO  1. TACGTCACCGTCCAGCACAAG  1 TATGTGACGGTGCAACATAA 2  2. CTCAACTACATCCTGCTCAAC  3 CTGAATTATATTTTATTGAAT  4 3. CAGCTCGTCTTCACCGTCAAG  5 CAATTGGTGTTTACGGTGAAA  6 4. ATCTATATCATGATGAACAAG  7 ATTTACATTATGATGAATAAA  8 5. GCCTACATGTTTCTGCTGATC  9 GCTTATATGTTCTTATTAATT 10 6. TACATGTTTCTGCTGATCGTG 11 TATATGTTCTTATTAATTGTC 12 7. CTGCGCACGCCTCTCAACTAC 13 TTACGGACCCCCTTGAATTAT 14 8. CGCACGCCTCTCAACTACATC 15 CGGACCCCCTTGAATTATATT 16 9. CTCAAGCCGGAGGTCAACAAC 17 TTGAAACCCGAAGTGAATAAT 1810. CAGCTCGTCTTCACCGTCA 19 CAATTGGTGTTTACGGTGA 2011. TACGCCAGCGTGGCATTCTAC 21 TATGCTTCTGTCGCCTTTTAC 2212. CCAGCGTTCTTTGCCAAGA 23 CCCGCCTTTTTCGCTAAAA 2413. GTCATCTATATCATGATGAAC 25 GTGATTTACATTATGATGAAT 2614. AACTGCATGCTCACCACCATC 27 AATTGTATGTTGACGACGATT 2815. ACCATCTGCTGCGGCAAGA 29 ACGATTTGTTGTGGGAAAA 3016. GACGATGAGGCCTCTGCTA 31 GAGGACGAAGCTAGCGCCA 3217. CACCTCTCTGCATGGATACT 33 CACGAGCTTACACGGGTATT 34 siRNAs Targeting 5′UTR 18. AGCTCAGGCCTTCGCAGCA 35 19. CAGGCCTTCGCAGCATTCT 36siRNAs Targeting 3′ UTR 20. TCACTTTCTTCTCCTATAA 3721. TAGTTAATGTTGTGAATAA 38 22. GCTCCTATGTTGGTATTAA 3923. AGTCACATAGGCTCCTTAA 40 24. GATTCTTGCTTTCTGGAAA 4125. ACAGTAGGTGCTTAATAAA 42 26. GAACATATCTATCCTCTCA 4327. CTGTACAGATTCTAGTTAA 44 28. TGTGAATAACATCAATTAA 4529. CAATTAATGTAACTAGTTA 46 30. TGATTATCACCTCCTGATA 4731. GCAGTCATCAGACCTGAAA 48 32. TGTCATCCTTACTCGAAGA 4933. GAATTAAGCTGCCTCAGTA 50 34. GCCAGAAGCTCTAGCTTTA 5135. AGCTCTGCCTGGAGACTAA 52 siRNAs Targeting an Intron36. GATCTTATTTGGAGCAATA 53 37. TGGCTGTGATCCAGGAATA 5438. GATGCATTCTTCTGCTAAA 55 39. GCAATATGCGCTTGTCTAA 5640. TTGTCTAATTTCACAGCAA 57 41.TGTTTGTTGCATTCAATAA 5842. CCAGAGCGCTAAGCAAATA 59 43. GTCTTGCATTTAACAGGAA 6044. GGCTGTGATCCAGGAATAT 61 45. TGCAGGAGGAGACGCTAGA 6246. CTTTCACTGTTAGGAATGT 63 47. TTTGGTTGATTAACTATAT 6448. TTAACTATATGGCCACTCT 65 49. AGATGTTCGAATTCCATCA 66siRNAs Targeting a Polymorphism 50. TCTTCACCGTCAAGGAGGTAT 67TGTTTACGGTGAAAGAAGTAC 68

siRNA sequences 1-17 target the human rhodopsin coding sequence. siRNAsequences 18 and 19 target the human rhodopsin 5′UTR. siRNA sequences20-35 target the human rhodopsin 3′UTR. siRNA sequences 36-49 targethuman rhodopsin intronic sequence. The sequence of the sense strand ofthe siRNA is given. Notably, siRNAs may also target a combination ofthese. For example, an siRNA target site may be in the 5′UTR and exon 1.Or an siRNA target site may be in the coding region and an intron. Or ansiRNA target site may be in an exon and the 3′UTR. siRNA sequence 50 isan example of an siRNA that has a target site that spans Exon 3/intron 3of the human rhodopsin gene. The site contains a known polymorphism inintron 3. If this site was used as an siRNA target, the replacement genewould have the wildtype base at the polymorphic site but degeneracy ofthe genetic code could be used to change other bases at the replacementsite. The siRNA(s) may comprise all or part of the sequence provided.The sequences of replacement human rhodopsin nucleic acids over thetarget for siRNA-mediated suppression are provided for siRNA sequences1-17. Replacement nucleic acids include at least one alterednucleotide(s) at degenerate position(s) over the siRNA target site(highlighted in bold print). Thus, replacement sequences here provideone of multiple replacement options. Some replacement constructs containnucleotide changes in the coding sequence. These replacement constructswhile altered in nucleotide sequence encode the same amino acids as thewild type rhodopsin protein. Other replacement constructs are altered ateither silent or non-silent polymorphic sites. These replacementconstructs encode wild type protein, with wild type function. For siRNAstargeting the UTRs or intronic sequence, no replacement constructs havebeen suggested because the number of base changes within the site is notlimited to degenerate positions (as is the case for sequence coding foramino acids).

It is notable that suppression of a given gene such as rhodopsin may beevaluated in a variety of animal species. The siRNA sequences providedin Table 6B represent examples of RNAi sequences that are homologousbetween porcine and human rhodopsin. In some transgenic animal modelsthe presence of the human transgene enables direct evaluation ofsequences that target the human gene in that animal model. In otherinstances suppressor sequences may be chosen to maximise the homologybetween the human gene (for example, rhodopsin) and the endogenous genein the animal under evaluation.

TABLE 6B Exemplary siRNA Sequences Targeting HomologousSequences Between Human and Porcine Rhodopsin SEQ Suppression IDPosition in levels in siRNA Sequence NO: NM_000539.2 HeLa Cells P1ACCTCTCTGCATG 414 384-403 69% GATAGT-TT P2 CATGTTCGTGGTC 415 713-732 81%CACTTC-TT

siRNA can be expressed in miR vectors using polymerase II promoters. Forthis purpose pcDNA6.2-GW/EmGFP-miR from Invitrogen is used where thecloned miR-155 gene is recombined in order to express the choice ofsiRNA. The antisense strand of the siRNA is kept intact followed by amodified terminal loop and the sense strand, which is modified byintroducing a deletion of 2 central nucleotides in order to form aninternal loop. See Catalogue no K4936-00, Block-IT, POLII, miR RNAiexpression vector kits catalogue, Invitrogen, page 7 for figure showingthe native miR-155 sequence and the converted sequence of siRNA-lacZ inthe form of miR-lacZ.

Exemplary miRNA Sequences Targeting Human Rhodopsin:

CC miRNA oligos: Top strand: (SEQ ID NO: 416) 5′- TGCTGCTTCTTGTGCTGGACGGTGACGTTTTGGCCACTGACTG ACGTCACCGTAGCACAAGAAG - 3′Bottom strand: (SEQ ID NO: 417) 5′- CCTGCTTCTTGTGCTACGGTGACGTCAGTCAGTGGCCAAAACG TCACCGTCCAGCACAAGAAGC - 3′Q1 miRNA oligos: Top strand: (SEQ ID NO: 418) 5′- TGCTGGTAGTAGTCGATTCCACACGAGTTTTGGCCACTGACTG ACTCGTGTGGTCGACTACTAC - 3′Bottom strand: (SEQ ID NO: 419) 5′- CCTGGTAGTAGTCGACCACACGAGTCAGTCAGTGGCCAAAACT CGTGTGGAATCGACTACTACC - 3′BB miRNA oligos: Top strand: (SEQ ID NO: 420) 5′- TGCTGGTAGAGCGTGAGGAAGTTGATGTTTTGGCCACTGACTG ACATCAACTTTCACGCTCTAC - 3′Bottom strand: (SEQ ID NO: 421) 5′- CCTGGTAGAGCGTGAAAGTTGATGTCAGTCAGTGGCCAAAACA TCAACTTCCTCACGCTCTACC - 3′

In an embodiment of the invention, suppression agents and replacementgenes are expressed in photoreceptor cells to alleviate diseasepathology. In a further embodiment, replacement nucleic acids encode agene which when mutated may cause retinal degeneration other thanretinitis pigmentosa, for example, Stargarts Syndrome, glaucoma, cod-roddystrophy, corneal dystrophy or Age-related Macular Degeneration (AMD)(Table 5).

In another aspect, the invention provides cells expressing a suppressioneffector such as a dsRNA, either transiently or stably, for experimentalor therapeutic use. In an embodiment, the cells express an siRNA thattargets rhodopsin. In another embodiment, the cells express areplacement nucleic acid expressing rhodopsin that is not targeted bythe siRNA. In another embodiment, the cells comprise a vector encodingat least one or more siRNAs. In another embodiment, the cells comprise avector encoding a replacement nucleic acid. In an additional embodiment,the cells comprise one or more vectors encoding siRNA(s) and replacementnucleic acid(s).

In another aspect, the invention provides transgenic animals and theirexperimental or therapeutic use. In an embodiment, the transgenic animalis a model for Retinitis Pigmentosa, for example, an animal with amutation observed in humans such as the Pro23His and or Pro347sermutations. In another embodiment, the transgenic animal expresses adsRNA that targets human rhodopsin. In another embodiment, thetransgenic animal expresses a replacement nucleic acid transgene thathas been altered at one or more wobble position(s) such that it escapessuppression.

Suppression agents and replacement nucleic acids of the invention can beadministered to cells, tissues, plants and/or animals, either separatelyor together. In yet another aspect administration of suppression agentand/or replacement nucleic acid may be systemic or local. In yet anotheraspect, administration of suppression agent and replacement nucleic acidmay be used in conjunction with chemical and/or physical agents to aidadministration. In another aspect, the invention provides methods forsuppressing rhodopsin expression in an animal by intraocular (e.g.,subretinal or intravitreal) injection of a suppression agent into theanimal. In another aspect intraocular administration (e.g., subretinalinjection, intravitreal) is used to administer a suppression agentand/or replacement nucleic acid to an animal. In another embodiment,ionthophoresis or electroporation is used to administer suppressionagents and/or replacement nucleic acids. In another embodiment,suppression agents and/or replacement nucleic acids are administeredusing nanotechnology (Kawasaki and Player Nanomedicine 1(2):101-9, 2005;Silva Surg. Neurol. 67(2):113-6, 2007; Andrieu-Solar et al., Mol. Vis.12:1334-47, 2006) or bacteria (Daudel et al., Expert Rev. Vaccines6(1):97-110, 2007).

Suppression agents and replacement nucleic acids may be optimallycombined with conserved regions A-I and/or transcription factor bindingsites identified within conserved regions A-I and/or with enhancerelements and/or other regulatory elements (see Tables 1 and 2 above andTables 9-12 below).

In one aspect of the invention, there is provided a vector forexpression of a suppression agent for a disease causing gene and/or areplacement nucleic acid that is not recognized by the suppressionagent, wherein the vector comprises at least one of the conservedregions selected from: conserved region B from the rhodopsin generepresented by SEQ ID NO: 93, or a variant or equivalent thereof;conserved region C from the rhodopsin gene represented by SEQ ID NO: 94,or a variant or equivalent thereof; conserved region F and G from therhodopsin gene represented by SEQ ID NO: 97 or a variant or equivalentthereof; and conserved region A from the rhodopsin gene represented bySEQ ID NO: 92, or a variant or equivalent thereof. In a particularembodiment, the vector comprises at least one of the conserved regionsselected from: conserved region B from the rhodopsin gene represented bySEQ ID NO: 93, conserved region C from the rhodopsin gene represented bySEQ ID NO: 94, conserved region F and G from the rhodopsin generepresented by SEQ ID NO: 97; and conserved region A from the rhodopsingene represented by SEQ ID NO: 92.

In one embodiment of the invention the use of suppression andreplacement constructs in combination with one or more factors tofacilitate cell survival, cell viability and/or cell functioning iscontemplated. In relation to neurons, a range of neurotrophic and/orneuroprotective factors may be used inter alia brain derivedneurotrophic factor (BDNF), glial derived neurotrophic factor (GDNF),neurturin, ciliary derived neurotrophic factor (CNTF), nerve growthfactor (NGF), fibroblast growth factors (FGF), insulin-like growthfactors (IGF), pigment epithelium-derived factor (PEDG), hepatocytegrowth factor (HGF), thyrotrophin releasing hormone (TRH) and rodderived cone viability factor (RDCVF) amongst others. There issubstantial evidence in the literature that such factors may increasecell viability and/or cell survival for a range of cell types. Forexample, these factors have been shown to provide beneficial effects toa wide range of neuronal cell types including, for example,photoreceptors, when delivered either in protein or DNA forms (Buch etal., Mol. Ther., 2006; 14(5):700-709). The use of GDNF to augmentgene-based therapies for recessive disease has been demonstrated in mice(Buch et al., Mol. Ther., 2006; 14(5):700-709). Genes encodingneurotrophic/neuroprotective factors may be expressed from generalpromoters such as the CBA promoter (Buch et al., Mol. Ther., 2006;14(5):700-709) or from tissue specific promoters. Sequences to optimiseexpression of neurotrophic/neuroprotective factors such as thosesequences identified in Tables 1, 2, 9-13 may be included in constructs.

Sequences of a number of exemplary neurotrophic factors are provided inFIG. 17. DNA encoding one or more neurotrophic and/or neuroprotectivefactors may be utilised in conjunction with suppression and replacement.FIG. 18 provides examples of constructs incorporating suppression andreplacement sequences together with sequence encoding a factor promotingcell viability and/or cell functioning such as GDNF, neurturin, CNTFand/or RDCVF amongst others as described above. Well established artknown methods involving DNA restriction digestion, DNA ligation intoplasmids, bacterial transformation, characterization of transfectedbacterial colonies, plasmid purification and DNA sequencing may be usedto clone suppression and replacement and neuroprotection/neurotrophicsequences into DNA-based vectors. Examples of the design of suchconstructs are provided in FIG. 18

Constructs incorporating suppression and replacement andneurotrophic/neuroprotective factor(s) may be delivered using viraland/or non-viral vectors using art known methods (Andrieu-Soler et al.,Mil. Vis., 2006; 12:1334-47). Naked DNA, lipids, polymers,nanoparticles, electrotransfer amongst other methods have been used toachieve gene/nucleotide delivery in cells and animals. For example,lentiviral vectors and/or adenoassociated viral (AAV) vectors may beused to deliver constructs incorporating the 3 components defined above(suppression, replacement and neurotrophism/neuroprotection).3-component constructs in some instances may require vectors that havesignificant capacity in terms of size of DNA inserts. Many viral andnon-viral vectors have been characterised that can facilitate large DNAfragments including inter alia lentiviral vectors and some ofadenoassociated viral serotypes. For example, AAV serotype 2 capsid 5vectors (AAV2/5) have been shown to accommodate 8-9 kilobases of DNA(Alberto Aurrichio; British Society of Gene Therapy, 2008). One or morecomponents (suppression, replacement, neurotrophism/neuroprotection)may, for example, in the case of AAV be cloned between the AAV ITRS andor one or more components may be cloned into the backbone of the plasmidused to generate AAV. FIG. 18 provides key elements of the constructdesign (B). Utilisation of backbone plasmid sequences to carrycomponents of a multi-component construct can be used to optimise thepopulation of AAV vectors generated using that plasmid. Moreover, inrelation to eye disease, it is notable that there is significantevidence that AAV2/5 transduces photoreceptor efficiently. Generation ofAAV vectors carrying suppression and replacement sequences inconjunction with sequences encoding neurotrophic and/or neuroprotectiveagents is contemplated. While AAV may be of value as a vector to deliver3-component constructs for some target tissues, a range of additionalviral and non-viral vectors are available for this purpose, such asthose described above, and vectors that are well know in the art.

While utilisation of a single vector to deliver 3-component constructsinvolving suppression and replacement and a neurotrophic/neuroprotectivesequence to a cell, a tissue and or an animal is contemplated, the useof multiple vectors in combination to deliver all 3 components is alsocontemplated. The multivalent approach involving suppression,replacement and neuroprotection may involve the use of 1 or more vectorsfor delivery. In addition, the 3 components may be delivered using acombination of a vector or vectors incorporating DNA sequences togetherwith RNA and or dsRNA and or protein. In the current invention, deliveryof protein, of RNA encoding protein and/or of DNA encoding protein or acombination thereof to achieve delivery of all 3 components,suppression, replacement and neuroprotection, is contemplated.

In another embodiment of the invention the size of the backbone of theAAV plasmid vector is either increased or decreased so as to increaseexpression from the virus. For example, it has been described in the artthat increasing the AAV virus backbone in size such that it is largerthan the insert cloned within ITSI and ITS2 favours AAV packaging of theinsert over packaging of the backbone, thereby increasing expression ofDNA cloned within the ITR regions (Bennet et al., Reversal of visualdefects in animal models of LCA within weeks of treatment with anoptimized AAV. Molecular Therapy Vol. 15, supplement 1, s286).

In a further embodiment of the invention the size of the backbone isincreased with a gene which is therapeutically beneficial driven by apromoter. In this embodiment a portion of packaged AAV consists of thebackbone and hence a portion of AAV particles will express the geneencoded within the backbone. In one embodiment the therapeuticallybeneficial gene cloned in the backbone is a neurotrophic factor suchGDNF, Neurturin or others.

While the invention can be used for dominant and or polygenic disorders,it may also be practised for recessive disorders. For example, the artdescribes that when treating the recessive disorder phenylketonuria(PKU) with replacement genes, endogenous protein expressed from mutantgenes interfered with protein from replacement genes (Described in athesis submitted to the University of Florida in partial fulfillment ofthe requirements for the degree of Doctor of Philosophy, by CatherineElisabeth Charron, August 2005 and entitled “Gene therapy forphenylketonuria: dominant-negative interference in a recessivedisease”). Thus, suppression and replacement constructs may be targetedto recessive disorders which like PKU require suppression andreplacement.

Suppression and replacement technology provides a strategy that may beapplicable to a wide range of genetic disorders including disorderscharacterized by either a recessive, dominant, polygenic, multifactorialor a dominant negative pathology. In a further embodiment of theinvention conserved regions identified in the promoter region ofmammalian rhodopsin genes and/or enhancer elements and/or otherregulatory elements and/or epigenetic elements such as listed in Table 5may be combined with suppressors targeting genes with mutations otherthan rhodopsin and providing replacement genes other than rhodopsin.Osteogenesis imperfecta, epidermolysis bullosa, autosomal dominant earlyonset Alzheimer's disease, autosomal dominant polycystic kidney disease,Rett syndrome, familial platelet disorder, dominant negative diabetesinsipidus, autosomal dominant Stargardt like macular dystrophy, nemalinemyopathy, familial pulmonary arterial hypertension, APC and p53 relatedcancers and several other disorders (OMIM) may potentially benefit froma suppression and replacement therapeutic approach. Triplet repeatdisorders, 14 of which have been characterised to date, includingHuntington's disease, spinocerebellar ataxia and myotonic dystrophy maybenefit from a suppression and replacement approach. For each disorder,promoters of the endogenous gene or constitutive promoters or promotesfrom other genes, or inducible promoters may be used to express thesuppression agent or replacement nucleic acid.

In another embodiment of the invention, promoter and/or enhancerelements and/or other regulatory elements and/or epigenetic elements maybe combined with other promoters than rhodopsin in combination withsuppression and/or replacement elements. For example, but notexclusively, promoter and enhancer elements can be combined with theCOL1A1 and or COL1A2 and or COL7A1 and or Keratin 5 and or Keratin 14and or peripherin and/or IMPDH1 promoters and/or genes. Depending uponthe tissue in which the suppression agent and/or replacement nucleicacid is administered or active in vivo, tissue specific regulatoryelements are used to enhance expression of the suppression agent and/orreplacement nucleic acid.

The suppressors and/or replacement nucleic acids of the invention can betargeted to suppress and replace a gene where mutations in the gene cangive rise, predispose or work in combination with other genetic factorsand/or environmental factors to cause disease pathology. For example, inthe case of dominant retinopathies the rhodopsin geen may be suppressedand replaced. For example, siRNAs targeting RHO- (NM_(—)000539.2) can bedesigned and provided commercially. Likewise control siRNAs, forexample, targeting EGFP (U57608) and or other reporter genes and orother non-targeting siRNAs can be designed and sythesised. siRNAs arechosen to target sequences which differed by at least one and preferablemany more nucleotides from any known gene in mouse and human databases(http://www.ncbi.nlm.nih.gov/blast, BLASTN2.2.6, Altschul et al., NucAcids Res. 25: (17:3389-402, 1997). siRNAs can be cloned downstream of,for example, polymerase III promoters such as the H1 or U6 promoters togenerate short hairpin RNAs (shRNAs; Brummelkamp et al., Science 296:(5567:505-3, 2001). Alternatively, polymerase II promoters which driveexpression in many or all cell or tissue types including the CMVpromoter, ubquitin promoter and or the β-actin promoter, for example,may be used to express shRNAs Likewise tissue specific promoters such asthe rhodopsin promoter, peripherin promoter and or enolase promoteramongst others may be used to express shRNAs. shRNA sequences can becloned into vectors with a reporter gene to facilitate monitoringexpression from vectors, for example, shRNAs can be cloned in pEGFP-1amongst other plasmids (BD Biosciences, Clontech, Palo Alto, Calif.).Suppressors can be delivered to cells, tissues and or animals with orwithout replacement nucleic acids.

Replacement nucleic acids with nucleotide sequence changes over thetarget site for siRNA-mediated suppression, for example, at degenerativenucleotides can be generated by primer directed mutagenesis and clonedinto vectors such as pcDNA3.1- (Invitrogen). Replacement nucleic acidsmay also be modified at the UTRs and or at polymorphic sites within thetarget gene. Ubiquitous promoters such as the CMV promoter and or theubiquitin promoter and or the β-actin promoter amongst others can beused to drive expression of replacement nucleic acids. Alternatively,tissue specific promoters such as the rhodopsin promoter, peripherinpromoter, Col1A1 promoter, Col1A2 promoter, Col1A7 promoter, Keratinpromoters and/or the enolase promoter amongst others and/or induciblepromoters such as a tetracycline responsive promoter can be used todrive expression of replacement nucleic acids. Replacement humanrhodopsin nucleic acids which have been altered in nucleotide sequenceat degenerate positions over siRNA target sites for example, replacementnucleic acids for siRNA sequences 1-17 are provided in Table 5.Replacement nucleic acids can be delivered to cells, tissues and oranimals with or without suppressor agents.

Suppression and Replacement in Cells and Tissues

Promoter driven replacement nucleic acids such as rhodopsin nucleicacids and siRNAs and/or shRNAs targeting rhodopsin can be co-transfectedinto cells, for example, HeLa and or Cos-7 cells amongst other celltypes using art known methods. For example, 24 hours post-transfectionof suppressor agents and/or replacement nucleic acids, RNA andcytoplasmic protein can be isolated from cells using well establishedmethodologies. Additionally, suppression and replacement can beevaluated in tissues. In the case of retinal genes, for example,organotypic retinal explant cultures from mouse or rat, for example, canbe prepared and maintained using art known methods and suppressor agentsand or replacement nucleic acids can be delivered to organotypiccultures. For example, electroporation can be used to deliver siRNAand/or shRNA constructs and/or shRNA constructs and replacement nucleicacids to retinal explants as described in Palfi et al., Hum. Mutat.27(3):260-8, 2006. Subsequent to electroporation of retinal explants,retinas can be treated with trypsin to expedite dissociation of cells.Retinal cell sub-populations within the dissociated cell populationwhich have a particular feature, for example, that express a reportergene such as EGFP can be identified. One method of identification thatcan be invoked is FACS (Palfi et al., Hum. Mutat. 27(3):260-8, 2006).Levels of suppression and replacement of a target gene can be evaluatedin FACS isolated cell populations. For example, suppression and/orsuppression and replacement can be evaluated in electroporated EGFPpositive cells from retinal explants.

Evaluation of Suppression and Replacement Using RNA Assays

Suppression and replacement can be evaluated in cells, tissues and/oranimals using RNA assays including real time RT-PCR, northern blotting,RNA in situ hybridisation and or RNAse protection assays. RNA expressionlevels of suppressors and/or of endogenous genes and or replacementnucleic acids can be assessed by real time RT-PCR using, for example, a7300 Real Time PCR System (Applied Biosystems, Foster City, Calif., USA)and using, for example, a QuantiTect SYBR Green RT-PCR kit (Qiagen Ltd).RT-PCR assays are undertaken using levels of expression of housekeepingcontrols such as β-actin or GAPDH, for example, for comparativepurposes. Levels of RNA expression can be evaluated using sets ofprimers targeting the nucleic acids of interest including suppressors,target genes and/or replacements, for example, the following primers canbe used for the evaluation of levels of expression of human rhodopsin,β-actin and GAPDH.

TABLE 7 PCR Primers for measuring rhodopsin, β-actin, and GAPDH SEQ IDPrimer Sequence NO RHO forward 5′ CTTTCCTGATCTGCTGGGTG 3′ 69 primerRHO reverse 5′ GGCAAAGAACGCTGGGATG 3′ 70 primer β-actin forward 5′TCACCCACACTGTGCCCATCTACGA 3′ 71 primer β-actin reverse 5′CAGCGGAACCGCTCATTGCCAATGG 3′ 72 primer GAPDH forward5′-CAGCCTCAAGATCATCAGCA-3′ 73 primer: GAPDH reverse5′-CATGAGTCCTTCCACGATAC-3′ 74 primer:

Expression of replacement constructs and/or shRNAs may be confirmed, forexample, by Northern blotting. RNA may also be detected by in situhybridisations using single stranded RNA probes that have been labelledwith, for example, DIG. To evaluate levels of expression of suppressionagents and/or replacement nucleic acids and/or endogenous target genes,RNase protections assays can be performed using art known methods, suchas that described in the Ambion mirVana™ Probe and Marker kit manual(catalogue number 1554) and the Ambion RPAIII™ Ribonuclease protectionassay kit manual (catalogue number 1414). For example, RNA probesapproximately 15-25 nucleotides in length specific for transcripts from,for example, an endogenous target gene and/or a suppressor and/or areplacement nucleic acid can be synthesized. For example, RNA probestargeting mouse rhodopsin and/or human rhodopsin and/or suppressionagents targeting rhodopsin and/or rhodopsin replacement nucleic acidscan be synthesized using companies such as Sigma-Proligo or Ambion. RNAprobes and size standards can be labelled to aid visualization afterseparation of samples on denaturing polyacrylamide gels. For example,RNA probes and Decade™ size marker (Ambion Inc) can be 5′ end-labelledwith P³²-γATP (GE Healthcare) using the mirVana™ probe and marker kitaccording to the manufacturer's protocol (Ambion Inc.). RNase protectionassays can be performed using art known methods, for example, using theRPA III™ Ribonuclease Protection Assay Kit and the manufacturer'sprotocol (Ambion Inc.). Expression of suppressors and/or replacementnucleic acids and/or endogenous genes can be undertaken and determinedin cells, in tissues and or in animals using, for example, the assaysand associated methodologies provided above.

Evaluation of Suppression and Replacement Using Protein Assays

Suppression and replacement can be evaluated in cells, tissues and/oranimals using protein assays including ELISA, western blotting andimmunocytochemistry assays. ELISAs can be undertaken to evaluate levelsof suppression by assessing levels of expression of a target endogenousgene and/or can be used to evaluate levels expression of replacementnucleic acids—such proteins assays are well know in the art and methodsare provided in, for example, Palfi et al., Hum. Mutat. 27(3):260-8,2006. For example, in the case of retinal genes such as the rhodopsingene, ELISA is undertaken using a rhodopsin primary antibody which istypically used in a diluted form, for example, using a 1/10-1/10000dilution (but possibly outside of this range) of an antibody for thetarget protein. In addition, Western Blotting may be undertaken todetermine relative quantities of a specific protein, for examplerhodopsin. Briefly, protein samples are separated using SDS-PAGE andtransferred to a membrane. The membrane is incubated with genericprotein (for example milk proteins) to bind to “sticky” places on themembrane. A primary antibody is added to a solution which is able tobind to its specific protein and a secondary antibody-enzyme conjugate,which recognizes the primary antibody is added to find locations wherethe primary antibody bound.

In addition to the protein assays referred to above, assays usingantibodies in conjunction with microscopy can be used to evaluateprotein levels. For example, in the case of rhodopsinimmunocytochemistry (for example, using a 1/10-1:1000 dilution of aprimary rhodopsin antibody) and fluorescent microscopy can be carriedout as has been documented in Kiang et al., 2005 Mol. Ther.12(3):555-61, 2005. Immunocytochemistry can be undertaken on cellsand/or tissues. In the case of the retina, various modes of sectioningcan be implemented to evaluate retinal sections. For example, frozensections, agar embedded sections and/or resin embedded sections can beused. To obtain thin sections, for example of the retina, epon embeddingand semi-thin sectioning can be performed using art known methods suchas those provided in McNally et al., Hum. Mol. Genet. 11(9):1005-16,2002. Immunocytochemistry may be used to evaluate suppression of atarget gene and or expression of replacement nucleic acids.Additionally, histological analyses can be used to evaluate thehistological effect(s) associated with the administration of suppressorsand or replacement nucleic acids. In animal models of retinaldegenerations such as the rho−/−, rds, rhodopsin Pro23H is, rhodopsinPro2347Ser mice and others there is a degeneration of the photoreceptorcell layer over time. Histological analyses can be used to evaluate ifthis degeneration has been modulated subsequent to administration ofsuppression agents and/or replacement nucleic acids.

Delivery of Suppression and Replacement

Both non-viral and/or viral vectors can be used in the invention todeliver the suppression agents and/or replacement nucleic acids. Forexample, in the case of retina, recombinant adenoassociated virus (AAV)and more specifically AAV2/5 has previously been found to elicitefficient transduction of photoreceptor cells. Other AAV serotypes mayalso be used to deliver to retina, for example, AAV2/2 elicits efficientdelivery to the retinal pigment epithelium (RPE) as does AAV4. AAVvectors can be generated using protocols with and without helper virus.For example, a helper virus free protocol using a triple transfectionapproach is well documented (Xiao et al., J. Virol. 72(3):2224-32,1998). Expression cassettes carrying suppression and/or replacementelements can be cloned into plasmids such as pAAV-MCS provided byStratagene Inc. Suppressors and/or replacement nucleic acids are clonedbetween the inverted terminal repeats of AAV2 and transfected into 293cells (Stratagene; ATACC cat no CRL-1573) with two other plasmids, hencethe term triple transfection. For example, the pRep2/Cap5 plasmid(Hildinger et al., J. Virol. 75(13):6199-203, 2001) together with thepHelper plasmid (Stratagene), at, for example, a ratio of 1:1:2, can beused to generate AAV2/5 vectors. Virus can be generated using a varietyof art known procedures including the method outlined below. Forexample, to generate virus fifty 150 mm plates of confluent HEK293 cellswere transfected (50 μg DNA/plate) with polyethyleminine (Reed et al.,J. Virol. Methods 138(1-2):85-98, 2006). 48 hrs post-transfection crudeviral lysates were cleared (Auricchio et al., 2001) and purified byCsCl₂ gradient centrifugation (Zolotukhin et al., Gene Ther.6(6):973-85, 1999). The AAV containing fraction was dialyzed againstPBS. Genomic titres, viral particles (vp/ml), were determined byquantitative real-time PCR using art known methods (Rohr et al., J.Virol. Methods 106(1):81-8, 2002). AAVs can be generated that contain,for example, either targeting shRNAs or control shRNAs and/orreplacement nucleic acids such as rhodopsin and/or reporter nucleicacids such as EGFP and/or stuffer sequences and/or sequences aidingexpression of suppression agents and/or replacement nucleic acids suchas promoter and/or enhancer sequences and/or other regulatory sequencesand/or epigenetic elements.

Administration of Suppression and Replacement Vectors

Animal models can be used to mirror human disorders. For example, animalmodels of human retinopathies or that express a human retinal gene havebeen generated, for example, rho−/− mice (Humphries et al., Nat. Genet.15(2):216-9, 1997), NHR+/− mice (Olsson et al., Neuron 9(5):815-30,1992), Pro23H is mice (Olsson et al., Neuron 9(5):815-30, 1992),Pro347Ser mice (Li et al., Proc. Natl. Acad. Sci. U.S.A. 95(20):11933-8,1998) and RHO-M mice (see below). Mice typically are maintained underspecific pathogen free (SPF) housing conditions and in a controlledlight environment. The suppression agents and/or replacement nucleicacids of the invention can be administered to animals either locallyand/or systemically. Local administration can include direct injectionto the target tissues and/or in the proximity of the target tissue ashas been described in detail in the art in, for example, Xia et al. (ACSChem. Biol. 1(3):176-83, 2004) delivered AAV vectors with shRNAs tobrain to treat spinocerebellar ataxia. In the case of the retina,subretinal injection can be used to administer suppression agents and/orreplacement nucleic acids according to the following procedure. Forexample, mice can be anaesthetised by intraperitoneal injection ofDomitor and Ketalar (10 and 50 μg/g of body weight respectively). Thepupils are dilated with phenylephrine and under local analgesia(amethocaine) a small puncture is made in the sclera. A micro-needleattached to a 10 μl syringe (Hamilton Company Europe) is insertedthrough the puncture to the subretinal space and 1-3 μl of vector isadministered. For example, in the case of AAV 1-3 μl of a 10¹²⁻¹⁴ vp/mlAAV vector preparation in PBS is administered. A reverse anaesthetic(antisedan, 50 μg/g of body weight) can be applied by intraperitonealinjection post-delivery. Body temperature during the procedure issustained using a homeothermic heating device. In addition newborn micecan be prepared for subretinal injection according to Matsuda and Cepko(Proc. Natl. Acad. Sci. U.S.A. 101(1):16-22, 2004).

Assay for Function

To evaluate if suppression and/or replacement modulates the function ofa target tissue and/or cell type, one or more assays may be employedthat are well described in the prior art. In the case of the retina,functional assays include but are not limited to electrophysiology, suchas pattern electroretinogram (ERG), full field ERG, and visual evokedpotentials. In addition, visual field assessments, color visionassessments, and pupilometry may be performed. For example,electroretinography can be used to evaluate the response of the retinato light. This can be performed using, for example, the followingprocedure or an adapted procedure. Animals can be dark-adapted overnightand prepared for ERG under dim red light. Pupils are dilated with 1%cyclopentalate and 2.5% phenylephrine. Animals are anesthetized withketamine and xylazine (16 and 1.6 μg/10 g body weight respectively)injected intraperitoneally. Standardized flashes of light are presentedto the animal, for example a mouse, in a Ganzfeld bowl. ERG responsesare recorded simultaneously from both eyes by means of contact lenselectrodes (Medical Workshop, Netherlands) using 1% amethocaine astopical anesthesia. Reference and ground electrodes are positionedsubcutaneously, approximately one mm from the temporal canthus andanterior to the tail respectively. Responses are analysed using aRetiScan RetiPort electrophysiology unit (Roland Consulting Gmbh). Theprotocol is based on that approved by the International ClinicalStandards Committee for human electroretinography. Rod-isolatedresponses are recorded using a dim white flash (−25 dB maximal intensitywhere maximal flash intensity was 3 candelas/m²/s) presented in thedark-adapted state. Maximal combined rod-cone responses to the maximalintensity flash are then recorded. Following a 10 minute lightadaptation to a background illumination of 30 candelas/m², cone-isolatedresponses are recorded to the maximal intensity flash presentedinitially as a single flash and subsequently as 10 Hz flickers. A-wavesare measured from the baseline to the trough and b-waves from thebaseline (in the case of rod-isolated responses) or from the a-wave tothe trough.

The agents of the invention are administered in effective amounts. Aneffective amount is a dosage of the agent sufficient to provide amedically desirable result. An effective amount means that amountnecessary to delay the onset of, inhibit the progression of or haltaltogether the onset or progression of the particular condition ordisease being treated. An effective amount may be an amount that reducesone or more signs or symptions of the disease. When administered to asubject, effective amounts will depend, of course, on the particularcondition being treated; the severity of the condition; individualpatient parameters including age, physical condition, size and weight,concurrent treatment, frequency of treatment, and the mode ofadministration. These factors are well known to those of ordinary skillin the art and can be addressed with no more than routineexperimentation.

Actual dosage levels of active ingredients in the pharmaceuticalcompositions of the invention can be varied to obtain an amount of theagent(s) that is effective to achieve the desired therapeutic responsefor a particular patient, compositions, and mode of administration. Theselected dosage level depends upon the activity of the particular agent,the route of administration, the severity of the condition beingtreated, the condition, and prior medical history of the patient beingtreated. However, it is within the skill of the art to start doses ofthe agent(s) at levels lower than required to achieve the desiredtherapeutic effort and to gradually increase the dosage until thedesired effect is achieved.

The agents and pharmaceutical compositions of the invention can beadministered to a subject by any suitable route. For example, thecompositions can be administered orally, including sublingually,rectally, parenterally, intracisternally, intravaginally,intraperitoneally, topically and transdermally (as by powders,ointments, or drops), bucally, or nasally. The term “parenteral”administration as used herein refers to modes of administration otherthan through the gastrointestinal tract, which include intravenous,intramuscular, intraperitoneal, intrasternal, intramammary, intraocular,retrobulbar, intrapulmonary, intrathecal, subcutaneous andintraarticular injection and infusion. Surgical implantation also iscontemplated, including, for example, embedding a composition of theinvention in the body such as, for example, in the brain, in theabdominal cavity, under the splenic capsule, brain, or in the cornea.

Agents of the present invention also can be administered in the form ofliposomes. As is known in the art, liposomes generally are derived fromphospholipids or other lipid substances. Liposomes are formed by mono-or multi-lamellar hydrated liquid crystals that are dispersed in anaqueous medium. Any nontoxic, physiologically acceptable, andmetabolizable lipid capable of forming liposomes can be used. Thepresent compositions in liposome form can contain, in addition to acompound of the present invention, stabilizers, preservatives,excipients, and the like. The preferred lipids are the phospholipids andthe phosphatidyl cholines (lecithins), both natural and synthetic.Methods to form liposomes are known in the art. See, for example,Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, NewYork, N.Y. (1976), p. 33, et seq.

Dosage forms for topical administration of an agent of this inventioninclude powders, sprays, ointments, and inhalants as described herein.The agent is mixed under sterile conditions with a pharmaceuticallyacceptable carrier and any needed preservatives, buffers, or propellantswhich may be required. Ophthalmic formulations, eye ointments, powders,and solutions also are contemplated as being within the scope of thisinvention.

Pharmaceutical compositions of the invention for parenteral injectioncomprise pharmaceutically acceptable sterile aqueous or nonaqueoussolutions, dispersions, suspensions, or emulsions, as well as sterilepowders for reconstitution into sterile injectable solutions ordispersions just prior to use. Examples of suitable aqueous andnonaqueous carriers, diluents, solvents, or vehicles include waterethanol, polyols (such as, glycerol, propylene glycol, polyethyleneglycol, and the like), and suitable mixtures thereof, vegetable oils(such, as olive oil), and injectable organic esters such as ethyloleate. Proper fluidity can be maintained, for example, by the use ofcoating materials such as lecithin, by the maintenance of the requiredparticle size in the case of dispersions, and by the use of surfactants.

These compositions also can contain adjuvants such as preservatives,wetting agents, emulsifying agents, and dispersing agents. Prevention ofthe action of microorganisms can be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It also may bedesirable to include isotonic agents such as sugars, sodium chloride,and the like. Prolonged absorption of the injectable pharmaceutical formcan be brought about by the inclusion of agents which delay absorption,such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of the agent, it isdesirable to slow the absorption of the drug from subcutaneous orintramuscular injection. This result can be accomplished by the use of aliquid suspension of crystalline or amorphous materials with poor watersolubility. The rate of absorption of the agent then depends upon itsrate of dissolution which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of a parenterallyadministered drug from is accomplished by dissolving or suspending theagent in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe agent in biodegradable polymers such a polylactide-polyglycolide.Depending upon the ratio of agent to polymer and the nature of theparticular polymer employed, the rate of agent release can becontrolled. Examples of other biodegradable polymers includepoly(orthoesters) and poly(anhydrides). Depot injectable formulationsalso are prepared by entrapping the drug in liposomes or microemulsionswhich are compatible with body tissue.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial- or viral-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable medium just prior to use.

The invention provides methods for oral administration of apharmaceutical composition of the invention. Oral solid dosage forms aredescribed generally in Remington's Pharmaceutical Sciences, 18th Ed.,1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89. Solid dosageforms for oral administration include capsules, tablets, pills, powders,troches or lozenges, cachets, pellets, and granules. Also, liposomal orproteinoid encapsulation can be used to formulate the presentcompositions (as, for example, proteinoid microspheres reported in U.S.Pat. No. 4,925,673). Liposomal encapsulation may include liposomes thatare derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556).In general, the formulation includes an agent of the invention and inertingredients which protect against degradation in the stomach and whichpermit release of the biologically active material in the intestine.

In such solid dosage forms, the agent is mixed with, or chemicallymodified to include, a least one inert, pharmaceutically acceptableexcipient or carrier. The excipient or carrier preferably permits (a)inhibition of proteolysis, and (b) uptake into the blood stream from thestomach or intestine. In a most preferred embodiment, the excipient orcarrier increases uptake of the agent, overall stability of the agentand/or circulation time of the agent in the body. Excipients andcarriers include, for example, sodium citrate or dicalcium phosphateand/or (a) fillers or extenders such as starches, lactose, sucrose,glucose, cellulose, modified dextrans, mannitol, and silicic acid, aswell as inorganic salts such as calcium triphosphate, magnesiumcarbonate and sodium chloride, and commercially available diluents suchas FAST-FLO®, EMDEX®, STA-RX 1500®, EMCOMPRESS® and AVICEL®, (b) binderssuch as, for example, methylcellulose ethylcellulose,hydroxypropyhnethyl cellulose, carboxymethylcellulose, gums (e.g.,alginates, acacia), gelatin, polyvinylpyrrolidone, and sucrose, (c)humectants, such as glycerol, (d) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, sodium carbonate, starch including the commercialdisintegrant based on starch, EXPLOTAB®, sodium starch glycolate,AMBERLITE®, sodium carboxymethylcellulose, ultramylopectin, gelatin,orange peel, carboxymethyl cellulose, natural sponge, bentonite,insoluble cationic exchange resins, and powdered gums such as agar,karaya or tragacanth; (e) solution retarding agents such a paraffm, (f)absorption accelerators, such as quaternary ammonium compounds and fattyacids including oleic acid, linoleic acid, and linolenic acid (g)wetting agents, such as, for example, cetyl alcohol and glycerolmonosterate, anionic detergent surfactants including sodium laurylsulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate,cationic detergents, such as benzalkonium chloride or benzethoniumchloride, nonionic detergents including lauromacrogol 400, polyoxyl 40stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60,glycerol monostearate, polysorbate 40, 60, 65, and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose; (h)absorbents, such as kaolin and bentonite clay, (i) lubricants, such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, polytetrafluoroethylene (PTFE), liquid paraffin,vegetable oils, waxes, CARBOWAX® 4000, CARBOWAX® 6000, magnesium laurylsulfate, and mixtures thereof; (j) glidants that improve the flowproperties of the drug during formulation and aid rearrangement duringcompression that include starch, talc, pyrogenic silica, and hydratedsilicoaluminate. In the case of capsules, tablets, and pills, the dosageform also can comprise buffering agents.

Solid compositions of a similar type also can be employed as fillers insoft and hard-filled gelatin capsules, using such excipients as lactoseor milk sugar, as well as high molecular weight polyethylene glycols andthe like.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells, such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They optionally can contain opacifying agents and also can be of acomposition that they release the active ingredients(s) only, orpreferentially, in a part of the intestinal tract, optionally, in adelayed manner. Exemplary materials include polymers having pH sensitivesolubility, such as the materials available as EUDRAGIT® Examples ofembedding compositions which can be used include polymeric substancesand waxes.

The active compounds also can be in micro-encapsulated form, ifappropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Inaddition to the active compounds, the liquid dosage forms can containinert diluents commonly used in the art, such as, for example, water orother solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol ethyl carbonate ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethyl formamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydroflirfurylalcohol, polyethylene glycols, fatty acid esters of sorbitan, andmixtures thereof.

Besides inert diluents, the oral compositions also can includeadjuvants, such as wetting agents, emulsifying and suspending agents,sweetening, coloring, flavoring, and perfuming agents. Oral compositionscan be formulated and further contain an edible product, such as abeverage.

Suspensions, in addition to the agent(s), can contain suspending agentssuch as, for example ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar, tragacanth, and mixtures thereof.

Also contemplated herein is pulmonary delivery of the agent(s) of theinvention. The agent(s) is delivered to the lungs of a mammal whileinhaling, thereby promoting the traversal of the lung epithelial liningto the blood stream. See, Adjei et al., Pharmaceutical Research7:565-569 (1990); Adjei et al., International Journal of Pharmaceutics63:135-144 (1990) (leuprolide acetate); Braquet et al., Journal ofCardiovascular Pharmacology 13 (suppl.5): s.143-146 (1989)(endothelin-1); Hubbard et al., Annals of Internal Medicine 3:206-212(1989) (α1-antitrypsin); Smith et al., J. Clin. Invest. 84:1145-1146(1989) (α1-proteinase); Oswein et al., “Aerosolization of Proteins,”Proceedings of Symposium on Respiratory Drug Delivery II, Keystone,Colo., March, 1990 (recombinant human growth hormone); Debs et al., TheJournal of Immunology 140:3482-3488 (1988) (interferon-γ and tumornecrosis factor α) and Platz et al., U.S. Pat. No. 5,284,656(granulocyte colony stimulating factor).

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including, but not limited to, nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art.

Some specific examples of commercially available devices suitable forthe practice of the invention are the ULTRAVENT® nebulizer, manufacturedby Mallinckrodt, Inc., St. Louis, Mo.; the ACORN II® nebulizer,manufactured by Marquest Medical Products, Englewood, Colo.; the VENTOL®metered dose inhaler, manufactured by Glaxo Inc., Research TrianglePark, N.C.; and the SPINHALER® powder inhaler, manufactured by FisonsCorp., Bedford, Mass.

All such devices require the use of formulations suitable for thedispensing of a agent(s) of the invention. Typically, each formulationis specific to the type of device employed and can involve the use of anappropriate propellant material, in addition to diluents, adjuvants,and/or carriers useful in therapy.

The composition is prepared in particulate form, preferably with anaverage particle size of less than 10 □m, and most preferably 0.5 to 5□m, for most effective delivery to the distal lung.

Carriers include carbohydrates such as trehalose, mannitol, xylitol,sucrose, lactose, and sorbitol. Other ingredients for use informulations may include lipids, such as DPPC, DOPE, DSPC and DOPC,natural or synthetic surfactants, polyethylene glycol (even apart fromits use in derivatizing the inhibitor itself), dextrans, such ascyclodextran, bile salts, and other related enhancers, cellulose andcellulose derivatives, and amino acids.

Also, the use of liposomes, microcapsules or microspheres, inclusioncomplexes, or other types of carriers is contemplated.

Formulations suitable for use with a nebulizer, either jet orultrasonic, typically comprise a compound of the invention dissolved inwater at a concentration of about 0.1 to 25 mg of biologically activeprotein per mL of solution. The formulation also can include a bufferand a simple sugar (e.g., for protein stabilization and regulation ofosmotic pressure). The nebulizer formulation also can contain asurfactant to reduce or prevent surface-induced aggregation of theinhibitor composition caused by atomization of the solution in formingthe aerosol.

Formulations for use with a metered-dose inhaler device generallycomprise a finely divided powder containing the agent suspended in apropellant with the aid of a surfactant. The propellant can be anyconventional material employed for this purpose, such as achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid also can be useful as a surfactant.

Formulations for dispensing from a powder inhaler device comprise afinely divided dry powder containing the agent and also can include abulking agent, such as lactose, sorbitol, sucrose, mannitol, trehalose,or xylitol, in amounts which facilitate dispersal of the powder from thedevice, e.g., 50 to 90% by weight of the formulation.

Nasal delivery of the agent(s) and composition of the invention also iscontemplated. Nasal delivery allows the passage of the agent orcomposition to the blood stream directly after administering thetherapeutic product to the nose, without the necessity for deposition ofthe product in the lung. Formulations for nasal delivery include thosewith dextran or cyclodextran. Delivery via transport across other mucousmembranes also is contemplated.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the agent(s) of theinvention with suitable nonirritating excipients or carriers, such ascocoa butter, polyethylene glycol, or suppository wax, which are solidat room temperature, but liquid at body temperature, and therefore meltin the rectum or vaginal cavity and release the active compound.

In order to facilitate delivery of the agent(s) across cell and/ornuclear membranes, compositions of relatively high hybrophobicity arepreferred. The agent(s) can be modified in a manner which increaseshydrophobicity, or the agent(s) can be encapsulated in hydrophobiccarriers or solutions which result in increased hydrophobicity.

Practice of the invention will be still more fully understood from thefollowing examples, which are presented herein for illustration only andshould not be construed as limiting the invention in any way.

EXEMPLIFICATION Example 1 siRNAs Targeting Human Rhodopsin and RhodopsinReplacement Nucleic Acids

siRNAs targeting human rhodopsin were synthesized and evaluated forRNAi-mediated suppression (listed in Table 8). Suppression andreplacement constructs with suppressors targeting the human rhodopsinmRNA sequence and replacement rhodopsin genes that escape suppression bythe suppressor due to subtle changes in the sequence were subsequentlydesigned. These changes, while enabling replacement nucleic acids toescape suppression at least in part, did not change the protein productexpressed from the replacement genes. Short hairpin RNAs (shRNAs) wereused to demonstrate suppression in vivo (FIG. 1). The sequence of thesense and antisense strands of the shRNAs is the same as the sequenceused for the siRNAs. An intervening loop is included between the senseand antisense strands in the same manner as Brummelkamp et al., Science296(5567):550-3, 2001. Notably, the number of nucleotides and the makeup of the nucleotides in the intervening loop can vary. The construct(s)were delivered using an AAV2/5 recombinant virus. Non-targeting siRNAcan be used as controls, for example, a non-targeting siRNA directedtowards an EGFP reporter gene can be used—for example.

siRNAs were designed according to the method of Elbashir et al., Nature411(6836):494-8, 2001, or by using the HiPerformance siRNA designalgorithm (Qiagen Ltd. Crawley, UK). siRNA target sequences differed byat least 4 nucleotides from any non-rhodopsin sequences in mouse andhuman databases (http://www.ncbi.nlm.nih.gov/blast, BLAST2.2.6 (Altschulet al., Nucleic Acids Res. 25(17):3389-402, 1997). siBB, siQ1 and anon-targeting siRNA siNT (5′ UUCUCCGAACGUGUCACGU 3′; SEQ ID NO:75) orEGFP (U57608), siEGFP (nt 256-277) were initially cloned downstream ofthe H1 promoter using BglII/BamH1 and Hind III restriction sites togenerate shRNAs and subsequently in pEGFP-1 (BD Biosciences, Clontech,Palo Alto, Calif.) using EcoRI and Hind III sites generating shBB-EGFP,shQ1-EGFP and shNT-EGFP (FIG. 1A). The EGFP gene enabled viraltransduction to be monitored. Six siRNAs sequences targeted the codingregion of human rhodopsin. Replacement nucleic acids were cloned intopCDNA3.1-plasmid (Invitrogen, Karlsruhe, Germany). The CMV promoter wasreplaced with either the human ubiquitin C promoter (pUB6/V5-His,Invitrogen) or a 1.7 kb fragment of the mouse rho promoter (rhoP).Sequence alterations were introduced into replacement nucleic acidsusing primer directed PCR-based mutagenesis using art known methods.Replacement nucleic acids with sequence alterations over the targetsites for siB, siBB, siC, siCC, siQ1 and siQ2 were termed rB, rBB, rC,rCC, rQ1, and rQ2. Altered nucleotides in the replacement rhodopsinsequences are at wobble positions (highlighted in bold print). Thesereplacement genes were designed to avoid suppression by the siRNAs yetencode wild type protein. Table 8 provides one replacement example foreach siRNA target site; however, in each case there are severalalternative possible replacement sequences because some amino acids haveas many as six codons and others have four or three codons.

TABLE 8 siRNA Sequence and Replacement Rhodopsin Sequence SEQReplacement SEQ ID rhodopsin ID Position in siRNA Sequence NO sequenceNO NM_000539.2 siB TCAACTTCCTCA 75 ATAAATTTTTTGACC 76 256-277 CGCTCTACTGTAT siBB TCACCGTCCAGC 77 CTGTATGTGACGGTG 78 254-274 ACAAGAA CAGCACsiC CGTGTGGAATCG 79 AGCTGCGGTATAGAT 80 270-292 ACTACTA TATTA siCCCGCTCAAGCCGG 81 ACCTTGAAACCCGAA 82 274-294 AGGTCAA GTGAA siQ1TCAACTTCCTCA 83 CTGTATGTGACGGTG 84 650-670 CGCTCTACGT CAGCAC siQ2CTCTACGTCACC 85 CTGTATGTGACGGTG 86 671-694 GTCCAGCACAA CAGCAC

Suppression of RHO in HeLa Cells

RNAi-mediated suppression of RHO was initially evaluated in HeLa cells.siRNAs targeting RHO were co-transfected with a CMV promoter-driven wildtype RHO. Transfections were carried out three times in quadruplicateusing lipofectamine 2000 to aid transfections (Gibco-BRL). Real timeRT-PCRs, performed on RNAs extracted from transfected cells 24 hourspost-transfection, demonstrated up to 87% suppression (p<0.01, FIG. 2A)(see Table 7 for primer sequences). siRNAs siBB, siCC and siQ1 wereselected for further analysis. Similar levels of rhodopsin proteinsuppression were quantified by ELISA (up to 88%, p<0.01, FIG. 2A) anddemonstrated by immunocytochemistry 24 hours post-transfection (FIG.2B). Subsequently, replacement RHO constructs, rBB, rCC and rQ1, weregenerated incorporating nucleotide changes at degenerate positions overthe target sites for siRNAs, siBB, siQ1 and siCC as described above andshown in Table 8. Transfections were performed three times inquadruplicate in HeLa cells according to art known methods as describedabove. Results indicated that replacement RHO constructs were notsuppressed by corresponding siRNAs, for example, rBB by siBB (FIG. 3).However, significant levels of suppression were obtained with othernon-corresponding siRNAs, for example siQ1 suppressed rBB and rCC (FIG.3).

Long Term Suppression of RHO in Retinal Explants

To provide long term RHO suppression, siBB and siQ1 were cloned asshRNAs into an EGFP expressing vector (shBB-EGFP and shQ1-EGFP, FIG.1A). Plasmids were electroporated into retinal explants from newbornNHR+/− rho−/− mice using the methods described in Matsuda and Cepko(2004) Proc. Natl. Acad. Sci. USA 101: 16-22. NHR+/− mice express a wildtype human RHO gene and display a wild type phenotype. Cells fromretinal explants (n=6) were dissociated two weeks post-electroporationand EGFP-positive cells isolated by FACS (FIG. 4A). Real time RT-PCR wasperformed on RNA extracted from EGFP-positive FACS-isolated cells usingthe primers described in Table 8 and results obtained in explantsmirrored those found in HeLa cells. Results indicated that RHOsuppression of greater than 85% was achieved (p<0.001, FIG. 4B).

Long Term Suppression Using AAV Vectors

Long-term expression of therapies will be required for a progressiveretinopathy such as adRP. To achieve long-term suppression in vivo,shBB-EGFP and the non-targeting shNT-EGFP were engineered into AAVvectors (AAV-shBB-EGFP and AAV-shNT-EGFP) (FIG. 1A). Recombinant AAV2/5viruses were generated using a helper virus free system. Expressioncassettes were cloned into pAAV-MCS (Stratagene, La Jolla, Calif., USA),between the inverted terminal repeats of AAV2, and transfected intoHEK-293 cells (ATCC no. CRL-1573) with pRep2/Cap5 and pHelper(Stratagene), at a ratio of 1:1:2. Fifty 150 mm plates of confluentcells were transfected (50 μg DNA/plate) with polyethyleminine. Fortyeight hours post-transfection crude viral lysates were cleared andpurified by CsCl₂ gradient centrifugation. AAV-containing fractions weredialysed against PBS. Genomic titres, i.e., viral particles (vp/ml),were determined by quantitative real time PCR. AAVs generated containedthe shBB-EGFP and shNT-EGFP constructs (AAV-shBB-EGFP and AAV-shNT-EGFP,FIG. 1A).

The EGFP gene enabled viral transduction to be monitored. Three μl ofAAV-shBB-EGFP (2×1012 vp/ml) or AAV-shNT-EGFP (3×1012 vp/ml) weresubretinally injected into adult NHR+/− rho−/− mice. Two weekspost-injection two animals were sacrificed and expression of the 21nucleotide shBB shown in two retinas using RNase protection (FIG. 5A).Retinas were dissociated and EGFP-positive cells collected by FACS.RNAi-mediated suppression of RHO, as evaluated by real time RT-PCR (seeTable 8 for primer sequences) two weeks post-injection (n=6), wasapproximately 90% (p<0.001) in AAV-shBB-EGFP-transduced photoreceptorcells (FIG. 5B). Four retinas were dissociated and significantsuppression of rhodopsin protein expression was demonstrated in vivo inEGFP-positive transduced cells by immunocytochemistry (FIG. 5C).

Suppression in Transgenic Animals

A transgenic mouse expressing a sequence-modified RHO gene was generated(RHO-M). RHO-M+/− rho−/− were evaluated at two months of age for rescueof the retinal pathology present in rho−/− mice by histology (FIG. 6A-C)and ERG (FIG. 6D). Rhodopsin immunolabeling in rod outer segments andthe thickness of outer nuclear layers were similar in wild typerho+/+(FIG. 6A), NHR+/− rho−/− (FIG. 6B) and RHO-M+/− rho−/− (FIG. 6C)mice. Additionally, ERG responses were similar in wild type rho+/+,rho+/−, NHR+/− rho−/− and RHO-M+/− rho−/− mice. ERG b-waves ofrod-isolated responses of 500-700 μV were observed in mice of allgenotypes (FIG. 6D). The amplitudes and timings of the combined rod andcone responses to the maximal intensity flash presented in thedark-adapted state, as well as the light adapted cone-isolated responsesboth to single flash and 10 Hz flickers, were equivalent in all thegenotypes examined (data not shown). These results validate the use ofthe degeneracy of the genetic code to engineer codon-modified human RHOgenes which can provide functional human rhodopsin protein.

AAV-Delivered Suppression and Replacement of Human RHO In Vivo

Having established shBB and shQ1 as potent suppressors and rBB and rQ1as being refractory to their corresponding suppressors, shBB-rBB andshQ1-rQ1 were cloned into AAV vectors using the triple plasmid systemdetailed above and viruses containing both elements of the therapeuticswere generated (AAV-shBB-rBB (also termed AAV-BB8) and AAV-shQ1-rQ1(also termed AAV-Q1)) using the method detailed above. Three μl ofAAV-shBB-rBB was subretinally injected into adult wild type rho+/+ mice(n=12) and replacement RHO mRNA expression confirmed by RT-PCR and RNaseprotection using RNA extracted 10 days post-injection (data not shown).To demonstrate that AAV-delivered rBB is translated into protein, 2 μlof a 1:1 mix of AAV-shBB-rBB and AAV-CMV-EGFP was subretinally injectedinto 10 day old rho−/− mice (n=6). Two weeks post-injection rhodopsinand EGFP protein expression were determined using fluorescentmicroscopy. Marked rhodopsin expression, overlapping with EGFP, wasobserved in transduced areas (FIG. 7).

Subsequently, 1 μl of AAV-shBB-rBB or AAV-shQ1-rQ1 was subretinallyinjected into newborn Pro23His+/− rho+/− mice (n=10) that present with aretinal degeneration resulting in complete loss of photoreceptors by twoweeks of age. In all animals one eye was injected with therapeutic virus(either AAV-shBB-rBB or AAV-shQ1-rQ1) and the other with a control virus(AAV-EGFP). The early onset and rapid nature of the retinopathy in youngPro23H is pups precluded use of ERG as a readout for benefit. However,at ten days of age retinal histology was evaluated in semi-thin resinembedded sections cut at approximately 50 μm intervals throughout thecentral meridian of the eye (n=10). From each section approximately 40measurements of ONL thickness were taken. Since only a part of theretina is transduced by a single subretinal injection of AAV(particularly in newborn pups), to identify the transduced area ONLmeasurements were ordered by thickness and the 15% highest and lowestvalues grouped for analysis. Lowest values represent thinnest ONLreadings, most likely corresponding to peripheral areas of the retinaand thus not in close proximity to injection sites. Highest valuesrepresent thickest ONL readings, most likely corresponding to centralareas of the retina and thus in closer proximity to injection sites.Significant differences in ONL thickness between AAV-shBB-rBB- andAAV-EGFP-treated eyes were observed. The ONL of treated eyes was foundto be approximately 33% (p<0.001) thicker than control injectedcounterparts for the highest value groupings (FIG. 8A-C). In the lowestvalue groupings a difference of approximately 10% was observed (FIG.8A). These data provide evidence at the histological level thatAAV2/5-delivered RNAi in conjunction with provision of a codon-modifiedreplacement gene can beneficially modulate the retinopathy inPro23His+/− rho−/− mice.

RNAi-mediated suppression was evaluated in retinal tissue aftersub-retinal injection of AAV vectors expressing either a suppressortargeting rhodopsin (AAV-shBB-EGFP, AAV-shCC-EGFP and AAV-shQ1-EGFP) ora non-targeting control (AAV-shNT-EGFP). Mice expressing a humanrhodopsin replacement gene (referred to as RHO-M mice and detailed inthe section on suppression in transgenic animals) were subretinallyinjected with AAV vectors (AAV2/5), containing shRNA sequences for BB,CC and Q1 and an EGFP reporter gene (AAV-shBB-EGFP, AAV-shCC-EGFP andAAV-shQ1-EGFP). The presence of the EGFP reporter gene enabled isolationof the population of retinal cells that are EGFP positive and thereforehave received AAV using FACS to isolate these cell populations.AAV-delivered RNAi-mediated suppression with each suppressor (BB, CC andQ1) was evaluated using real-time RT-PCR in cell populationscharacterised by FACS and was compared to suppression obtained using AAVwith non-targeting control shRNA sequences (AAV-shNT-EGFP). Significantrhodopsin suppression was obtained with BB and Q1 suppressors, however,significantly lower levels of suppression were obtained with the CCsuppressor (FIG. 6E). The replacement gene in RHO-M mice was partiallyprotected from suppression due to the presence of two nucleotidemismatches between the CC suppressor sequence and the target site forsuppression in the human rhodopsin replacement gene. The replacementgene is partially protected from siRNA CC-based suppression by theintroduction of two nucleotide changes at degenerate sites in thereplacement gene. FIG. 6F illustrates depression of the ERG response inRHO-M eyes that have received AAV-shBB-EGFP (panel 1) or AAV-shQ1-EGFP(panel 2) when compared to eyes subretinally injected withAAV-shNT-EGFP. The top tracing in each panel represents the right eyewhich received the targeting AAV-shRNA vector and the bottom tracing ineach panel represents the left eye which received the controlnon-targeting AAV-shNT vector. In contrast no reduction/depression ofthe ERG was observed in RHO-M mice subretinally injected withAAV-shCC-EGFP (panel 3) vector; this is likely due to the reduced levelsof rhodopsin suppression observed with AAV-shCC-EGFP (see FIG. 6Eabove).

Example 2 Optimization of Expression of Suppression Agents andReplacement Nucleic Acids

Expression of suppression and/or replacement vectors was optimized byincluding in the vectors sequences that enhanced and/or modulateexpression levels at the RNA and/or protein level. A list of exemplarysequence elements is provided in Table 1, however, the enhancing and/ormodulating elements of the invention are not exclusive to this list. Forexample, one or more of a promoter, a stuffer, an insulator, a silencer,a chromatin remodelling sequence, an intron sequence, a poly adenylationsignal, a post translational regulatory element, and a transcriptionfactor binding site can be included in suppression and/or replacementconstructs to modulate expression of suppression and/or replacementcomponents relating to the invention. Such elements and derivativesthereof can be used to modulate levels of expression, tissuespecificity, timing of expression, and/or induction of expression. Table9 provides some exemplary sequences that can be used to modulateexpression of suppression and/or replacement constructs relating to theinvention. The sequences provided are within conserved regions asevaluated by comparison of sequences from multiple species. At any oneposition a nucleotide may not be conserved between all species—thesequences represent regions where overall there is a high degree ofconservation. Such conserved sequences from any species such as human,mouse, rat, bacteria, virus and/or indeed a hybrid sequence from morethan one species could be used in the invention.

TABLE 9 Exemplary Enhancer SequencesCMV enhancer element amplified from pCDNA3.1 Invitrogen nt308-734 http://www.invitrogen.com/ (SEQ ID NO: 87)CCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGT GTAT CATA TGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTG TTTTGGCACC AAAATCAACG GGACpAAV.BB11 The WPR element from pSin11 CMV GFPpre mut FL(Gene Therapy (7): 641-5 (2006)) (SEQ ID NO: 88)GAGCAT CTTACCGCCATTTATTCCCA TATTTGTTCT GTTTTTCTTG ATTTGGGTATACATTTAAATGTTAATAAAA CAAAATGGTG GGGCAATCAT TTACATTTTTAGGGATATGTAATTACTAGT TCAGGTGTAT TGCCACAAGA CAAACATGTTAAGAAACTTTCCCGTTATTT ACGCTCTGTT CCTGTTAATC AACCTCTGGATTACAAAATTTGTGAAAGAT TGACTGATAT TCTTAACTAT GTTGCTCCTTTTACGCTGTGTGGATATGCT GCTTTATAGC CTCTGTATCT AGCTATTGCTTCCCGTACGGCTTTCGTTTT CTCCTCCTTG TATAAATCCT GGTTGCTGTCTCTTTTAGAGGAGTTGTGGC CCGTTGTCCG TCAACGTGGC GTGGTGTGCTCTGTGTTTGCTGACGCAACC CCCACTGGCT GGGGCATTGC CACCACCTGTCAACTCCTTTCTGGGACTTT CGCTTTCCCC CTCCCGATCG CCACGGCAGAACTCATCGCCGCCTGCCTTG CCCGCTGCTG GACAGGGGCT AGGTTGCTGGGCACTGATAATTCCGTGGTG TTGTCpAAV.BB13 The WPR element from pBSK11 (Donello JE, et al. J.Virol. 1998 72(6): 5085-92.) (SEQ ID NO: 89)AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGC CGCCTCCCC(Wild type woodchuck hepatitis B virus genome sequence ACCESSION J04514)

Example 3 Comparison of Rhodopsin Genes

In addition to adding enhancing and/or modulating elements tosuppression and/or replacement vectors, the rhodopsin promoter wasstudied in detail. A comparison of rhodopsin genes present in differentmammals resulted in identification of 9 highly conserved regions in therhodopsin gene (conserved regions A though I, Sequence 1, Table 10).Regions A, B, C and D are in the rhodopsin promoter region, conservedregion E is in intron 2 of the gene and conserved regions F, G, H and Iare in the 3′ region.

The following sequence (Sequence 1; Table 10) shows the conservedregions within the mouse promoter human intronic and exonic and 3′sequence. Notably, conserved sequences in the mouse promoter are nearlythe same in the human rhodopsin promoter and it is contemplated that thehuman or other mammalian rhodopsin promoters and/or derivatives and/orhybrids thereof may be used in suppression and replacement constructs.Additionally, it is contemplated that other promoters could be combinedwith some or all of conserved regions A though I and used in suppressionand/or replacement constructs, for example, other retinal promotersequences may be used.

TABLE 10 Conserved Regions of RhodopsinSequence 1: Mouse rhodopsin promoter sequence (upper case) ending at the Xho Isite (highlighted in bold print), followed by the human rhodopsin 5′UTR, humanrhodopsin exons and introns and human rhodopsin 3′region sequence (lower case).Conserved regions A - I are highlighted in bold print. (SEQ ID NO: 90).GTTCCAGGGC CCAGGGGCTT CCAGCCATGA GGGCACCTAG ACTTGTAATCCCTAGAGTCC TCCTGATGCC ACTGCCCAGG GACAGACAGC ACACAGCACCCCTCCCCCAC TCTCTTAACA GGCAGAAGCA GGGAGATGGA GGCATGCTGAAGATGTCCAT GTGAGGCTGG TGGTAGCATG CCCACTGCTG GGATGAAGAGATGGGGGCAA AGTGAGTGGC AGAGGCCAGG CCAGGTCCAG GCCCTTCCAGGCTTCCTCTG CCACTGTGGA GATGAAAGAG GGAGCCAGGC AAGGTCCAGGCCCTCCCCAC CCCCTCTGCC TCTATGGAGA TGAAGGGGGA ATGAAGAAGGGAGCCAGACA GTTGTGCCAA CACAACTCCT CCGTCGAGTG TCTAATTGCTTATGATCATG CATGCTCTCT CTCCCACTAA ACATTTATTA ATGTGTTAGG cons reg AATTTCCATTA GCGCGTGCCT TGAACTGAAA TCATTTGCAT ATGGCTGGGAAAAAGTGGGG TGAGGGAGGA AACAGTGCCA GCTCCCCAAC AGGCGTCAATCACAGTGACA GATCAGATGG TTTCTGGCTG GAGGCAGGGG GGCTGTCTGAGATGGCGGCA TGCATCCTTT CAGTGCATAT CACAGAAATT CAGGTGACTCCTGCTGGGAG CCAAGACCCT GAGGCTGAGC CTGGCCACAG CTCCAATAGCTGCTGGATAT CATCATGTCT GGGCTGAGCA GCCTCTAGAG GTACCCTTTTACAGATAGTA AAACTGAGGC TCAGTGACTG CTGAGCCAAA GTTGGACCCACCCACACTCA TTTGCAGACT GCCGTGGGCC ATGTTCTGAT CTCTTCCCTACCTGGACTCA GCCCAGCACA CTCGGCACAC AAGGCCCTTC TTCAGCTTGAATACAGCGTC CTCAGCTATA GCCAGCATCT ATGAATGGAG CTCAGTGACCCTGACTGGAG GAAGTTAGGA CAGGGATTTT TTCTGGAGTT TTGGCAGGAAGAGGCCAGGG TCAGGTGACT GCTGGAGCAC ACAGCTTGGT AAGACTAGTCAGGACCTGCG TCCTGAGGCT ACATGTCATA TCCACAGTAA GGAAGTGGAAGATGGGAGAT GACTGGCTGG GCCACAACCA GTGAGTGGAA TGTCCTTGTGCATCTTTGTT TCCTAACCTT CCCCTCTGTA GCTGCTGAAA CACACACACACCCCATGCTC TGTTATGCCT CTTCCCTGGC CTGGGATTTC CATGGCTGAGGTGATGGGGC ACTGAGGCAC CGCCAGGAAA GGCTGTAACC CATCTGCTCCCCCATCCTTC ACCAGACTTC AAGCACCTAC CTAGAGCACA GGTGCAATTTTGTACCCTCC CTGTCTGGGA CCCACAGTGG TTCCTCAATG CCGGCCAACCAGACTCATAG GCCTGCCCAC AAGGCCCTTG GGGCTATCTG TCTGAGGCCTGCAGGTGCCC TCCTGGCCAC CTAGGCTCCT GTGAGACTTA GACTTCCATAGATTCTTCCT GAAAGACTAC TGAGGGCAGG AGCCCCCAAG CCTCAGGGTTAGCTTTCCTC AGCCCTGCCT CTTTGCTAGC TCCGTTTCCA CATTGAAGGCAGGGCTGAGC AGGGCAGGCG CAGCGAGGAG CTAACTGCTG CTTCTCTCTCGTTCATTTGT CTGCTGCCCT GAGACGCCAC AGCACCTAAT AAGAGCATGTTATGTGTAGC AAACATTAGG CCTGTAAGGA AGGAAAGGAG TGACGTCCCTTGACGTCCTC AGCTAGGCTG TGGTGACACA AGCAAGAGGA CTAAGCCACAGGTGAGGAGA AAGGGGGGGG GGGGTCTGCT GACCCAGCAA CACTCTTTCC cons reg BTTCTGAGGCT TAAGAGCTAT TAGCGTAGGT GACTCAGTCC CTAATCCTCCATTCAATGCC CTGTGACTGC CCCTGCTTCT GAAGGGCCAA CATGGCTACAGCTAGCTCCA GAGACAGCTT TTCAGGGCCC CAGCATCCAA GCATCTCACAGTTCTCCACT GACCACACTC CTGTGCAGCA CTGGGCTTTT CAATGCCCCTGACTTGAAGA GAACTCAAAC TGCAGGTCAA CTAGACTCTG CAAACTTCACCTGTGCTGGG GGTTCCTAGC CTGTGGGGAC AGTGTATCTT GAATACCTGCTGCTATGGAC CAAGAGCTGA ACACACAGAC AAACAGGCTC AGCTGGCCGGCATTCTGGAA CCACAAATGA GTGTGGATGA GCAGGAGGGC AACAAAATGGTCTGGGTGTT GTCAACACAG TCAGTAAACA ATGCACGCAG TGGGGCTGGGCCCTGATGTG GAGCTAGGTG GGGTTGGCTC TCCTTGGAAA CCTGAAGGGAGAAGGAGAGG GAGCGAGATG ATGAGGTTTA TCAGCCTGCA GAGGCAGGGGGTCAGGAAGG AGTGCCACTG TACTGACCCA GGACCTCTGT GGGACATCAAGCCATGCCAA GGAGCCATGG AGCCTCGATT GCACTGGCAG GGACAGGTTGTGATGCCCCA GAGTCCCCAG ACCCAGCAAA CAGAGGCCCA GAGTGGGAAGTGGAGCTTTC CAGGGTATCG GGGTGACTCA GAGACACAGG GTAGAATCTGCCTTGGGTGC TCACTGCCCT ATCTGAGTCC ACATGGCTCA GTCCCCAGGCCCTGTTCTCT AGTGACTGTT GCTTTGATGA GGTAGAGACA GGCAGCCCTCTTCTAAGAAC TATGTTTTGA TGGGGGACTC AGAGTTGGGG TGGGGTGGCAATGAAATTCT GTAGACTGTG TGGTTATAAC CCTGGCTGTT ACTAGCTAGTTCTGTGACCT TGGTGACCCA CTTCAGACTC TAGGCCTCAG CCTCTGTAAGTGCAGATACA CAGCGCCAAT CAGCCGATGA CTTCTAACAA TACTCTTAACTCACACAGAG CTTGTCTCAC TGAGCCAACA CCCTGTACCC TCAGCTCAGTGACGGCTTTC AACCTGTGGG GCTGCCTCTG TTACCCAAGT GAGAGAGGGCCAGTGCTCCC AGAGGTGACC TTGTTTGCCC ATTCTCTCCC TGGGTCAGCCAGTGTTTATC TGTTGTATAC CCAGTCCACC CTGCAGGCTC ACATCAGAGCCTAGGAGATG GCTAGTGTCC CCGCGGAGAC CACGATGAAG CTTCCCAGCT HindIIIGTCTCAAGCA CAAGCTGGCT GCAGAGGCTG CTGAGGCACT GCTAGCTGGG start 1.734 kbGATGGGGGCA GGGTAGATCT GGGGCTGACC ACCAGGGTCA GAATCAGAACCTCCACCTTG ACCTCATTAA CGCTGGTCTT AATCACCAAG CCAAGCTCCT cons reg CTAAACTGCTA GTGGCCAACT CCCAGGCCCT GACACACATA CCTGCCCTGTGTTCCCAAAC AAGACACCTG CATGGAAGGA AGGGGGTTGC TTTTCTAAGCAAACATCTAG GAATCCCGGG TGCAGTGTGA GGAGACTAGG CGAGGGAGTACTTTAAGGGC CTCAAGGCTC AGAGAGGAAT ACTTCTTCCC TGGTTAGCCTCGTGCCTAGG CTCCAGGGTC TTTGTCCTGC CTGGATACCT ATGTGGCAAGGGGCATAGCA TTTCCCCCAC CATCAGCTCT TAGCTCAACC TTATCTTCTCGGAAAGACTG CGCAGTGTAA CAACACAGCA GAGACTTTTC TTTTGTCCCCTGTCTACCCC TGTAACTGCT ACTCAGAAGC ATCTTTCTCA CAGGGTACTGGCTTCTTGCA TCCAGAGTTT TTTGTCTCCC TCGGGCCCCC AGAATCAAATTCTTCCTCTG GGACTCAGTG GATGTTTCAC ACACGTATCG GCCTGACAGTCATCCTGGAG CATCCTACAC AGGGGCCATC ACAGCTGCAT GTCAGAAATGCTGGCCTCAC ATCCTCAGAC ACCAGGCCTA GTGCTGGTCT TCCTCAGACTGGCGTCCCCA GCAGGCCAGT AGGATCATCT TTTAGCCTAC AGAGTTCTGAAGCCTCAGAG CCCCAGGTCC CTGGTCATCT TCTCTGCCCC TGAGATTTTTCCAAGTTGTA TGCCTTCTAG GTAAGGCAAA ACTTCTTACG CCCCTCCTCGTGGCCTCCAG GCCCCACATG CTCACCTGAA TAACCTGGCA GCCTGCTCCCTCATGCAGGG ACCACGTCCT GCTGCACCCA GCAGGCCATC CCGTCTCCATAGCCCATGGT CATCCCTCCC TGGACAGGAA TGTGTCTCCT CCCCGGGCTGAGTCTTGCTC AAGCTAGAAG CACTCCGAAC AGGGTTATGG GCGCCTCCTCCATCTCCCAA GTGGCTGGCT TATGAATGTT TAATGTACAT GTGAGTGAACAAATTCCAAT TGAACGCAAC AAATAGTTAT CGAGCCGCTG AGCCGGGGGGCGGGGGGTGT GAGACTGGAG GCGATGGACG GAGCTGACGG CACACACAGCTCAGATCTGT CAAGTGAGCC ATTGTCAGGG CTTGGGGACT GGATAAGTCAGGGGGTCTCC TGGGAAGAGA TGGGATAGGT GAGTTCAGGA GGAGACATTGTCAACTGGAG CCATGTGGAG AAGTGAATTT AGGGCCCAAA GGTTCCAGTCGCAGCCTGAG GCCACCAGAC TGACATGGGG AGGAATTCCC AGAGGACTCTGGGGCAGACA AGATGAGACA CCCTTTCCTT TCTTTACCTA AGGGCCTCCACCCGATGTCA CCTTGGCCCC TCTGCAAGCC AATTAGGCCC CGGTGGCAGCAGTGGGATTA GCGTTAGTAT GATATCTCGC GGATGCTGAA TCAGCCTCTGGCTTAGGGAG AGAAGGTCAC TTTATAAGGG TCTGGGGGGG GTCAGTGCCTGGAGTTGCGC TGTGGGAGCC GTCAGTGGCT GAGCTCGCCA AGCAGCCTTGGTCTCTGTCT ACGAAGAGCC CGTGGGGCAG CCTCGAG XhoIggatcctgag tacctctcct ccctgacctc aggcttcctc ctagtgtcac cttggcccct conserved region Dcttagaagcc aattaggccc tcagtttctg cagcggggat taatatgatt atgaacacccccaatctccc agatgctgat tcagccagga gcttaggagg gggaggtcac tttataagggtctggggggg tcagaaccca gagtcatcca gctggagccc tgagtggctg agctcaggccttcgcagcat tcttgggtgg gagcagccac gggtcagcca caagggccac agccatgaatggcacagaag gccctaactt ctacgtgccc ttctccaatg cgacgggtgt ggtacgcagccccttcgagt acccacagta ctacctggct gagccatggc agttctccat gctggccgcctacatgtttc tgctgatcgt gctgggcttc cccatcaact tcctcacgct ctacgtcaccgtccagcaca agaagctgcg cacgcctctc aactacatcc tgctcaacct agccgtggctgacctcttca tggtcctagg tggcttcacc agcaccctct acacctctct gcatggatacttcgtcttcg ggcccacagg atgcaatttg gagggcttct ttgccaccct gggcggtatgagccgggtgt gggtggggtg tgcaggagcc cgggagcatg gaggggtctg ggagagtcccgggcttggcg gtggtggctg agaggccttc tcccttctcc tgtcctgtca atgttatccaaagccctcat atattcagtc aacaaacacc attcatggtg atagccgggc tgctgtttgtgcagggctgg cactgaacac tgccttgatc ttatttggag caatatgcgc ttgtctaatttcacagcaag aaaactgagc tgaggctcaa aggccaagtc aagcccctgc tggggcgtcacacagggacg ggtgcagagt tgagttggaa gcccgcatct atctcgggcc atgtttgcagcaccaagcct ctgtttccct tggagcagct gtgctgagtc agacccaggc tgggcactgagggagagctg ggcaagccag accectectc tctgggggcc caagctcagg gtgggaagtggattttccat tctccagtca ttgggtcttc cctgtgctgg gcaatgggct cggtcccctctggcatcctc tgcctcccct ctcagcccct gtcctcaggt gcccctccag cctccctgccgcgttccaag tctcctggtg ttgagaaccg caagcagccg ctctgaagca gttcctttttgctttagaat aatgtcttgc atttaacagg aaaacagatg gggtgctgca gggataacagatcccactta acagagagga aaactgaggc agggagaggg gaagagactc atttagggatgtggccaggc agcaacaaga gcctaggtct cctggctgtg atccaggaat atctctgctgagatgcagga ggagacgcta gaagcagcca ttgcaaagct gggtgacggg gagagcttaccgccagccac aagcgtctct ctgccagcct tgccctgtct cccccatgtc caggctgctgcctcggtccc attctcaggg aatctctggc cattgttggg tgtttgttgc attcaataatcacagatcac tcagttctgg ccagaaggtg ggtgtgccac ttacgggtgg ttgttctctgcagggtcagt cccagtttac aaatattgtc cctttcactg ttaggaatgt cccagtttggttgattaact atatggccac tctccctatg aaacttcatg gggtggtgag caggacagatgttcgaattc catcatttcc ttcttcttcc tctgggcaaa acattgcaca ttgcttcatggctcctagga gaggccccca catgtccggg ttatttcatt tcccgagaag ggagagggaggaaggactgc caattctggg tttccaccac ctctgcattc cttcccaaca aggaactctgccccacatta ggatgcattc ttctgctaaa cacacacaca cacacacaca cacacaacacacacacacac acacacacac acacacacac aaaactccct accgggttcc cagttcaatcctgaccccct gatctgattc gtgtccctta tgggcccaga gcgctaagca aataacttcccccattccct ggaatttctt tgcccagctc tcctcagcgt gtggtccctc tgccccttccccctcctccc agcaccaagc tctctccttc cccaaggcct cctcaaatcc ctctcccactcctggttgcc ttcctagcta ccctctccct gtctaggggg gagtgcaccc tccttaggcagtggggtctg tgctgaccgc ctgctgactg ccttgcaggt gaaattgccc tgtggtccttggtggtcctg gccatcgagc ggtacgtggt ggtgtgtaag cccatgagca acttccgcttcggggagaac catgccatca tgggcgttgc cttcacctgg gtcatggcgc tggcctgcgccgcaccccca ctcgccggct ggtccaggta atggcactga gcagaaggga agaagctccgggggctcttt gtagggtcct ccagtcagga ctcaaaccca gtagtgtctg gttccaggcactgaccttgt atgtctcctg gcccaaatgc ccactcaggg taggggtgta gggcagaagaagaaacagac tctaatgttg ctacaagggc tggtcccatc tcctgagccc catgtcaaac conserved region Eagaatccaag acatcccaac ccttcacctt ggctgtgccc ctaatcctca actaagctaggcgcaaattc caatcctctt tggtctagta ccccgggggc agccccctct aaccttgggcctcagcagca ggggaggcca caccttccta gtgcaggtgg ccatattgtg gccccttggaactgggtccc actcagcctc taggcgattg tctcctaatg gggctgagat gagactcagtggggacagtg gtttggacaa taggactggt gactctggtc cccagaggcc tcatgtccctctgtctccag aaaattccca ctctcacttc cctttcctcc tcagtcttgc tagggtccatttctacccct tgctgaattt gagcccaccc cctggacttt ttccccatct tctccaatctggcctagttc tatcctctgg aagcagagcc gctggacgct ctgggtttcc tgaggcccgtccactgtcac caatatcagg aaccattgcc acgtcctaat gacgtgcgct ggaagcctctagtttccaga agctgcacaa agatccctta gatactctgt gtgtccatct ttggcctggaaaatactctc accctggggc taggaagacc tcggtttgta caaacttcct caaatgcagagcctgagggc tctccccacc tcctcaccaa ccctctgcgt ggcatagccc tagcctcagcgggcagtgga tgctggggct gggcatgcag ggagaggctg ggtggtgtca tctggtaacgcagccaccaa acaatgaagc gacactgatt ccacaaggtg catctgcatc cccatctgatccattccatc ctgtcaccca gccatgcaga cgtttatgat ccccttttcc agggagggaatgtgaagccc cagaaagggc cagcgctcgg cagccacctt ggctgttccc aagtccctcacaggcagggt ctccctacct gcctgtcctc aggtacatcc ccgagggcct gcagtgctcgtgtggaatcg actactacac gctcaagccg gaggtcaaca acgagtcttt tgtcatctacatgttcgtgg tccacttcac catccccatg attatcatct ttttctgcta tgggcagctcgtcttcaccg tcaaggaggt acgggccggg gggtgggcgg cctcacggct ctgagggtccagcccccagc atgcatctgc ggctcctgct ccctggagga gccatggtct ggacccgggtcccgtgtcct gcaggccgct gcccagcagc aggagtcagc caccacacag aaggcagagaaggaggtcac ccgcatggtc atcatcatgg tcatcgcttt cctgatctgc tgggtgccctacgccagcgt ggcattctac atcttcaccc accagggctc caacttcggt cccatcttcatgaccatccc agcgttcttt gccaagagcg ccgccatcta caaccctgtc atctatatcatgatgaacaa gcaggtgcct actgcgggtg ggagggcccc agtgccccag gccacaggcgctgcctgcca aggacaagct actcccaggg caggggaggg gctccatcag ggttactggcagcagtcttg ggtcagcagt cccaatgggg agtgtgtgag aaatgcagat tcctggccccactcagaact gctgaatctc agggtgggcc caggaacctg catttccagc aagccctccacaggtggctc agatgctcac tcaggtggga gaagctccag tcagctagtt ctggaagcccaatgtcaaag tcagaaggac ccaagtcggg aatgggatgg gccagtctcc ataaagctgaataaggagct aaaaagtctt attctgaggg gtaaaggggt aaagggttcc tcggagaggtacctccgagg ggtaaacagt tgggtaaaca gtctctgaag tcagctctgc cattttctagctgtatggcc ctgggcaagt caatttcctt ctctgtgctt tggtttcctc atccatagaaaggtagaaag ggcaaaacac caaactcttg gattacaaga gataatttac agaacacccttggcacacag agggcaccat gaaatgtcac gggtgacaca gcccccttgt gctcagtccctggcatctct aggggtgagg agcgtctgcc tagcaggttc ccaccaggaa gctggatttgagtggatggg gcgctggaat cgtgaggggc agaagcaggc aaagggtcgg ggcgaacctcactaacgtgc cagttccaag cacactgtgg gcagccctgg ccctgactca agcctcttgccttccagttc cggaactgca tgctcaccac catctgctgc ggcaagaacc cactgggtgacgatgaggcc tctgctaccg tgtccaagac ggagacgagc caggtggccc cggcctaagacctgcctagg actctgtggc cgactatagg cgtctcccat cccctacacc ttcccccagccacagccatc ccaccaggag cagcgcctgt gcagaatgaa cgaagtcaca taggctcctt conserved region Faatttttttt ttttttttaa gaaataatta atgaggctcc tcactcacct gggacagcctgagaagggac atccaccaag acctactgat ctggagtccc acgttcccca aggccagcgggatgtgtgcc cctcctcctc ccaactcatc tttcaggaac acgaggattc ttgctttctggaaaagtgtc ccagcttagg gataagtgtc tagcacagaa tggggcacac agtaggtgct conserved region Gtaataaatgc tggatggatg caggaaggaa tggaggaatg aatgggaagg gagaacatatctatcctctc agaccctcgc agcagcagca actcatactt ggctaatgat atggagcagttgtttttccc tccctgggcc tcactttctt ctcctataaa atggaaatcc cagatccctggtcctgccga cacgcagcta ctgagaagac caaaagaggt gtgtgtgtgt ctatgtgtgtgtttcagcac tttgtaaata gcaagaagct gtacagattc tagttaatgt tgtgaataacatcaattaat gtaactagtt aattactatg attatcacct cctgatagtg aacattttgagattgggcat tcagatgatg gggtttcacc caaccttggg gcaggttttt aaaaattagctaggcatcaa ggccagacca gggctggggg ttgggctgta ggcagggaca gtcacaggaatgcaggatgc agtcatcaga cctgaaaaaa caacactggg ggagggggac ggtgaaggccaagttcccaa tgagggtgag attgggcctg gggtctcacc cctagtgtgg ggccccaggtcccgtgcctc cccttcccaa tgtggcctat ggagagacag gcctttctct cagcctctggaagccacctg ctcttttgct ctagcacctg ggtcccagca tctagagcat ggagcctctagaagccatgc tcacccgccc acatttaatt aacagctgag tccctgatgt catccttact conserved region Hcgaagagctt agaaacaaag agtgggaaat tccactgggc ctaccttcct tggggatgttcatgggcccc agtttccagt ttcccttgcc agacaagccc atcttcagca gttgctagtccattctccat tctggagaat ctgctccaaa aagctggcca catctctgag gtgtcagaattaagctgcct cagtaactgc tcccccttct ccatataagc aaagccagaa gctctagctttacccagctc tgcctggaga ctaaggcaaa ttgggccatt aaaagctcag ctcctatgttggtattaacg gtggtgggtt ttgttgcttt cacactctat ccacaggata gattgaaact conserved region Igccagcttcc acctgatccc tgaccctggg atggctggat tgagcaatga gcagagccaagcagcacaga gtcccctggg gctagaggtg gaggaggcag tcctgggaat gggaaaaaccccaactttgg ggtcatagag gcacaggtaa cccataaaac tgcaaacaag ctt

Conserved regions A through I and some sequence flanking the regions (5′and 3′, were combined (Table 11, SEQ ID NO: 92 through SEQ ID NO: 99,Sequence 2). This sequence was analyzed using MatInspector ReleaseProfessional 7.4.1 to identify other regions that may be involved intranscriptional and/or translational control of rhodopsin geneexpression. (A small portion of the Matinspector results are presentedin Table 12). This table illustrates some sequences within conservedregions A through I that are thought to be involved in the transcriptionand/or translation and/or stability of rhodopsin. Some of thesesequences, such as the CRX binding element in conserved region D and theTATA box in region G are known in the art. Others, such as the CRXbinding region in E, are not. The complete set of results fromMatInspector are presented in Table 13. 302 putative transcriptionbinding sites and/or regulatory sequences were identified and some arehighlighted in bold. On the basis of the conserved nature of regions Athough I and the important transcription factor binding sites thought tobe located within these regions, the constructs in FIG. 9 weregenerated. Construct BB16 contains conserved regions A, B, C, D, F andG. In addition an artificial CRX-NRL element (below) was insertedbetween conserved regions A and B. The components of the artificialCRX-NRL enhancer element include the CRX motif from conserved region D,the CRX motif from conserved region E and NRL binding sites areunderlined.

(SEQ ID NO: 91) TTTCTGCAGCGGGGATTAATATGATTATGAACACCCCCAATCTCCCAGATGCTGATTCAGCCAGGAGGTACC

All these constructs contain transcription binding sites identifiedwithin conserved regions A though I.

Sequence 2: Conserved regions A through I in the rhodopsin gene arehighlighted in bold below. The nucleotides of these sequences and asmall section of 5′ and 3′ sequence surrounding conserved regions havebeen numbered 1-1600. This sequence was analysed with MatInspector andthe nucleotide numbering system of sequence 2 (1-1600) relates to thenucleotide numbering system in Table 13.

TABLE 11 (Conserved regions are in bold)Conserved region A 1-210 (SEQ ID NO: 92)CACAACTCCT CCGTCGAGTG TCTAATTGCT TATGATCATGCATGCTCTCT CTCCCACTAA ACATTTATTA ATGTGTTAGGATTTCCATTA GCGCGTGCCT TGAACTGAAA TCATTTGCATATGGCTGGGA AAAAGTGGGG TGAGGGAGGA AACAGTGCCAGCTCCCCAAC AGGCGTCAAT CACAGTGACA GATCAGATGG TTTCTGGCTG 210Conserved region B 210-310 (SEQ ID NO: 93)AAGGGGGGGG GGGGTCTGCT GACCCAGCAA CACTCTTTCCTTCTGAGGCT TAAGAGCTAT TAGCGTAGGT GACTCAGTCC CTAATCCTCC ATTCAATGCC 310Conserved region C 310-410 (SEQ ID NO: 94)GGGGCTGACC ACCAGGGTCA GAATCAGAAC CTCCACCTTGACCTCATTAA CGCTGGTCTT AATCACCAAG CCAAGCTCCT TAAACTGCTA GTGGCCAACT 410Conserved region D 410-690 (SEQ ID NO: 95)aggcttcctc ctagtgtcac cttggcccct cttagaagcc aattaggccc tcagtttctg cagcggggattaatatgatt atgaacaccc ccaatctccc agatgctgat tcagccagga gcttaggagg gggaggtcactttataaggg tctggggggg tcagaaccca gagtcatcca gctggagccc tgagtggctg agctcaggccttcgcagcat tcttgggtgg gagcagccac gggtcagcca caagggccac agccatgaat ggcacagaag690 Conserved region E 690-850 (SEQ ID NO: 96)tcctgagccc catgtcaaac agaatccaag acatcccaac ccttcacctt ggctgtgccc ctaatcctcaactaagctag gcgcaaattc caatcctctt tggtctagta ccccgggggc agccccctct aaccttgggcctcagcagca ggggaggcca 850Conserved regions F and G 850-1220 (SEQ ID NO: 97)cccctacacc ttcccccagc cacagccatc ccaccaggag cagcgcctgt gcagaatgaa cgaagtcacataggctcctt aatttttttt ttttttttaa gaaataatta atgaggctcc tcactcacct gggacagcctgagaagggac atccaccaag acctactgat ctggagtccc acgttcccca aggccagcgg gatgtgtgcccctcctcctc ccaactcatc tttcaggaac acgaggattc ttgctttctg gaaaagtgtc ccagcttagggataagtgtc tagcacagaa tggggcacac agtaggtgct taataaatgc tggatggatg caggaaggaatggaggaatg aatgggaagg 1220Conserved region H 1220 1230-1316 1330 (SEQ ID NO: 98)tctagagcat ggagcctcta gaagccatgc tcacccgccc acatttaatt aacagctgag tccctgatgtcatccttact cgaagagctt agaaacaaag agtgggaaat 1330Conserved region I 1330 1342-1425 1600 (SEQ ID NO: 99)gctctagctt tacccagctc tgcctggaga ctaaggcaaa ttgggccatt aaaagctcag ctcctatgttggtattaacg gtggtgggtt ttgttgcttt cacactctat ccacaggata gattgaaact gccagcttccacctgatccc tgaccctggg atggctggat tgagcaatga gcagagccaa gcagcacaga gtcccctggggctagaggtg gaggaggcag tcctgggaat gggaaaaacc ccaactttgg ggtcatagag 1600

TABLE 12 Conserved sequence motifs in Rhodopsin Conserved regionPosition Name B 288-304 CRX C 366-382 CRX D 470-486 CRX E 784-764 CRX G1172-1177 TATA box D 500-520 Neuron-restrictive silencer factor E794-814 Neuron-restrictive silencer factor E 831-851 Neuron-restrictivesilencer factor

TABLE 13 Putative Rhodopsin Transcription Regulatory Factors Sequence(red: ci- value >60 capitals: SEQ Further Core Matrix core IDFamily/matrix Information Opt. Position Str. sim. sim. sequence) NOV$PDX1/1SL1.01 Pancreatic and 0.82 14-34 (+) 1.000 0.860 tcgagtgtcTAA100 intestinal lim- Ttgcttatg homeodomain factor V$HOMF/MSX.01Homeodomain 0.97 18-30 (+) 1.000 0.995 gtgtcTAATtgc 101 proteins MSX-1 tand MSX-2 V$HOXF/GSH2.01 Homeodomain 0.95 19-35 (+) 1.000 0.975tgtcTAATtgctt 102 transcription atga factor Gsh-2 V$GABF/GAGA.01GAGA-Box 0.78 33-57 (−) 1.000 0.825 gtgggAGAGag 103 agcatgcatgatcaV$FKHD/FREAC2.0 Fork head related 0.84 52-68 (+) 1.000 0.884 tcccacTAAAc104 1 activator-2 atttat (FOXF2) V$HOXF/HOXC13. Homeodomain 0.91 58-74(−) 1.000 0.914 acattaaTAAAt 105 01 transcription gttta factor HOXC13V$NKXH/HMX2.02 Hmx2/Nkx5-2 0.82 58-72 (−) 0.750 0.835 attaatAAATgtt 106homeodomain ta transcription factor V$SATB/SATB1.01 Special AT-rich 0.9458-72 (−) 1.000 0.956 attAATAaatgtt 107 sequence-binding ta protein 1,predominantly expressed in thymocytes, binds to matrix attachmentregions (MARs) V$BRNF/BRN3.02 Brn-3, POU-IV 0.89 59-77 (−) 1.000 0.892aacacatTAATa 108 protein class aatgttt V$PDX1/PDX1.01 Pdx1 0.74 59-79(−) 1.000 0.744 ctaacacatTAA 109 (IDX1/IPF1) Taaatgttt pancreatic andintestinal homeodomain TF V$PIT1/PIT1.01 Pit1, GHF-1 0.84 61-73 (+)1.000 0.857 acatTTATtaatg 110 pituitary specific pou domaintranscription factor V$BRNF/BRN3.02 Brn-3, POU-IV 0.89 62-80 (+) 1.0000.893 catttatTAATgt 111 protein class gttagg V$LHXF/LMX1B.0 LIM- 0.9162-76 (−) 1.000 0.946 acacatTAATaa 112 1 homeodomain atg transcriptionfactor V$HOXH/MEIS1B_ Meis1b and 0.78 64-78 (−) 0.750 0.823 TAACacattaat113 HOXA9.01 Hoxa9 form aaa heterodimeric binding complexes ontarget DNA V$HOXF/HOX1- Hox-1.3, 0.82 65-81 (+) 1.000 0.826ttatTAATgtgtt 114 3.01 vertebrate agga homeobox protein V$OCT1/OCT1.04Octamer-binding 0.80 77-91 (−) 0.846 0.866 ctAATGgaaatc 115 factor 1 ctaV$HOXF/PHOX2.01 Phox2a (ARIX) 0.87 78-94 (−) 1.000 0.969 gcgcTAATgga 116and Phox2b aatcct V$AHRR/AHRARN Aryl hydrocarbon 0.92 83-107 (+) 1.0000.932 ttccattagcgCG 117 T.01 receptor/Arnt TGccttgaactg heterodimersV$MOKF/MOK2.02 Ribonucleopro- 0.98 85-105 (+) 1.000 0.988 ccattagcgcgtg118 tein associated CCTTgaac zinc finger protein MOK-2 (human)V$EBOX/MYCMA MYC-MAX 0.91 87-101 (−) 1.000 0.918 aaggcaCGCGc 119 X.03binding sites taat V$HESF/HELT.01 Hey-like bHLH- 0.91 87-101 (−) 1.0000.947 aaggCACGcgc 120 transcriptional taat repressor V$HOMF/EN1.01Homeobox 0.77 97-109 (+) 0.782 0.776 gccTTGAactg 121 protein engrailedaa (en-1) V$OCT1/OCT1.02 Octamer-binding 0.85 109-123 (−) 1.000 0.992catATGCaaatg 122 factor 1 att V$OCTP/OCT1P.01 Octamer-binding 0.86113-125 (−) 1.000 0.910 gccATATgcaa 123 factor 1, POU- atspecific domain V$AIRE/AIRE.01 Autoimmune 0.86 119-145 (+) 0.916 0.862atatggctgggaaa 124 regulator aagTGGGgtga gg V$RBPF/RBPJK.02 Mammalian0.94 122-136 (+) 1.000 0.941 tggcTGGGaaa 125 transcriptional aagtrepressor RBP- Jkappa/CBF1 V$RXRF/VDR_RX Bipartite binding 0.75 123-147(+) 0.812 0.760 ggctgggaaaaag 126 R.06 site of tgGGGTgaggg VDR/RXR aheterodimers: 4 spacer nucleotides between the two directly repeatedmotifs V$NKXH/HMX3.01 H6 0.89 127-141 (+) 1.000 0.910 gggaaaAAGTg 127homeodomain gggt HMX3/Nkx5.1 transcription factor V$CIZF/NMP4.01NMP4 (nuclear 0.97 128-138 (+) 1.000 0.998 ggAAAAagtgg 128matrix protein 4)/ CIZ (Cas- interacting zinc finger protein)V$EBOX/SREBP.01 Sterol regulatory 0.90 132-146 (−) 1.000 0.960cccTCACccca 129 element binding cttt protein 1 and 2 V$RXRF/VDR_RXVDR/RXR 0.86 134-158 (+) 1.000 0.878 agtggggtgagg 130 R.02 Vitamin DGAGGaaacagt receptor RXR gc heterodimer site V$ETSF/PU1.01Pu.1 (Pu120) Ets- 0.89 141-157 (+) 1.000 0.895 tgagggaGGAA 131like transcription acagtg factor identified in lymphoid B- cellsV$NFAT/NFAT.01 Nuclear factor of 0.95 145-155 (+) 1.000 0.989 ggaGGAAaca132 activated T-cells g V$AREB/AREB6.04 AREB6 (Atp1a1 0.98 146-158 (−)1.000 0.991 gcactGTTTcct 133 regulatory c element binding factor 6)V$COMP/COMP1.0 COMP1, 0.77 163-185 (−) 1.000 0.811 ctgtgATTGacg 134 1cooperates with cctgttgggga myogenic proteins in multicomponent complexV$PAX6/PAX6.01 Pax-6 paired 0.77 163-181 (−) 0.808 0.781 gaTTGAcgcct 135domain binding gttgggga site V$MYBL/CMYB.01 c-Myb, important 0.90165-177 (+) 1.000 0.945 ccCAACaggcg 136 in hematopoesis, tc cellularequivalent to avian myoblastosis virus oncogene v-myb V$CREB/CREB.02cAMP- 0.89 167-187 (−) 1.000 0.902 cactgtgatTGA 137 responsive Cgcctgttgelement binding protein V$WHZF/WHN.01 Winged helix 0.95 169-179 (−)1.000 0.955 ttgACGCctgt 138 protein, involved in hair keratinizationand thymus epithelium differentiation V$HOXC/PBX1.01 Homeo domain 0.78170-186 (−) 1.000 0.840 actgtGATTgac 139 factor Pbx-1 gcctgV$PBXC/PBX1_ME Binding site for a 0.77 170-186 (−) 1.000 0.875actgTGATtgac 140 IS1.02 Pbx1/Meis1 gcctg heterodimer V$AP1R/TCF11MATCF11/MafG 0.81 177-201 (+) 1.000 0.838 caatcacagTGA 141 FG.01heterodimers, Cagatcagatggt binding to subclass of AP1 sitesV$TALE/MEIS1.01 Binding site for 0.95 183-193 (−) 1.000 0.971atcTGTCactg 142 monomeric Meis1 homeodomain protein V$HOXH/MEIS1A_Meis1a and 0.77 186-200 (+) 1.000 0.770 TGACagatcag 143 HOXA9.01Hoxa9 form atgg heterodimeric binding complexes on target DNAV$GATA/GATA3.0 GATA-binding 0.91 187-199 (+) 1.000 0.950 gacAGATcaga 1442 factor 3 tg V$AP4R/TAL1BET Tal-1beta/E47 0.87 189-205 (+) 1.000 0.955cagatCAGAtg 145 AE47.01 heterodimer gtttct V$NEUR/NEUROG. Neurogenin 10.92 191-203 (−) 1.000 0.925 aaaCCATctgat 146 01 and 3 (ngn1/3) cbinding sites V$ZBPF/ZBP89.01 Zinc finger 0.93 205-227 (−) 1.000 0.966agacccccccCC 147 transcription CCcttcagcca factor ZBP-89V$ZBPF/ZNF219.01 Kruppel-like zinc 0.91 207-229 (−) 1.000 0.997gcagaccCCCC 148 finger protein ccccccttcagc 219 V$INSM/INSM1.01Zinc finger 0.90 209- (+) 1.000 0.914 tgaagGGGGgg 149 protein 221nc gginsulinoma- associated 1 (IA- 1) functions as a transcriptionalrepressor V$EKLF/KKLF.01 Kidney-enriched 0.91 210- (+) 1.000 0.934gaagggGGGG 150 kruppel-like 226nc gggggtc factor, KLF15 V$EGRF/WT1.01Wilms Tumor 0.92 211- (+) 0.837 0.945 aagggGGGGg 151 Suppressor 227ncggggtct V$SP1F/GC.01 GC box elements 0.88 211- (+) 0.819 0.897aagggGGGGg 152 225nc ggggt V$EKLF/KKLF.01 Kidney-enriched 0.91 212- (+)1.000 0.949 agggggGGGG 153 kruppel-like 228nc gggtctg factor, KLF15V$SP1F/GC.01 GC box elements 0.88 213- (+) 0.819 0.908 gggggGGGGg 154227nc ggtct V$EGRF/WT1.01 Wilms Tumor 0.92 214- (+) 0.837 0.932gggggGGGGg 155 Suppressor 230nc gtctgct V$GLIF/ZIC2.01 Zinc finger 0.89214-228 (−) 1.000 0.967 cagacccCCCC 156 transcription ccccfactor, Zic family member 2 (odd- paired homolog, Drosophila)V$MAZF/MAZR.01 MYC-associated 0.88 215- (+) 1.000 0.972 ggggggGGGG 157zinc finger 227nc tct protein related transcription factorV$AP1R/BACH2.01 Bach2 bound 0.89 221-245 (−) 0.813 0.897 gagtgttgcTGG158 TRE Gtcagcagacccc V$AP1R/VMAF.01 v-Maf 0.82 221-245 (+) 1.000 0.957ggggtctgcTGA 159 Cccagcaacactc V$XBBF/MIF1.01 MIBP-1/RFX1 0.76 225-243(−) 0.800 0.778 gtgttgctggGTC 160 complex Agcaga V$XBBF/RFX1.01X-box binding 0.89 227-245 (+) 1.000 0.907 tgctgacccaGC 161 protein RFX1AAcactc V$NFAT/NFAT.01 Nuclear factor of 0.95 243-253 (−) 1.000 0.971gaaGGAAaga 162 activated T-cells g V$NKXH/HMX2.01 Hmx2/Nkx5-2 0.83253-267 (−) 1.000 0.911 gctCTTAagcct 163 homeodomain cag transcriptionfactor V$PAX8/PAX8.01 PAX 2/5/8 0.88 254-266 (−) 0.800 0.901ctcTTAAgcctc 164 binding site a V$NKXH/HMX2.01 Hmx2/Nkx5-2 0.83 256-270(+) 1.000 0.931 aggCTTAagag 165 homeodomain ctat transcription factorV$HOXF/PHOX2.01 Phox2a (ARIX) 0.87 260-276 (−) 1.000 0.898 acgcTAATagc166 and Phox2b tcttaa V$CLOX/CDPCR3. Cut-like 0.73 266-284 (−) 0.8800.770 agtcacctacgcta 167 01 homeodomain ATAGc protein V$EGRF/NGFIC.01Nerve growth 0.80 269-285 (+) 1.000 0.855 attaGCGTaggt 168factor-induced gactc protein C V$AP1R/BACH2.01 Bach2 bound 0.89 271-295(−) 1.000 0.957 attagggacTGA 169 TRE Gtcacctacgcta V$CREB/TAXCRETax/CREB 0.71 274-294 (+) 1.000 0.744 cgtaggTGACtc 170 B.02 complexagtccctaa V$AP1F/AP1.01 Activator protein 0.94 278-288 (+) 0.904 0.967ggtgACTCagt 171 1 V$AP1F/AP1.03 Activator protein 0.94 278-288 (−) 1.0000.976 acTGAGtcacc 172 1 V$HOXF/CRX.01 Cone-rod B 0.94 288-304 (+) 1.0000.972 tcccTAATcctc 173 homeobox- cattc containing transcriptionfactor/otx-like homeobox gene V$SORY/HBP1.01 HMG box- 0.86 298-310 (−)1.000 0.905 ggcattgAATG 174 containing ga protein 1 V$IRFF/IRF7.01Interferon 0.86 329-347 (+) 0.936 0.865 caGAATcagaa 175regulatory factor cctccacc 7 (IRF-7) V$RORA/RORA1.0 RAR-related 0.93342-360 (−) 1.000 0.953 ttaatgaGGTCa 176 1 orphan receptor aggtggaalpha1 V$CSEN/DREAM.0 Downstream 0.95 344-354 (−) 1.000 0.960agGTCAaggtg 177 1 regulatory element- antagonist modulator, Ca2+-bindingprotein of the neuronal calcium sensors family that binds DRE(downstream regulatory element) sites as a tetramer V$E4FF/E4F.01GLI-Krueppel- 0.82 345-357 (−) 0.789 0.824 atgAGGTcaag 178 related gttranscription factor, regulator of adenovirus E4 promoter V$HOXF/BARX2.0Barx2, 0.95 347-363 (−) 1.000 0.980 gcgtTAATgag 179 1 homeobox gtcaagtranscription factor that preferentially binds to paired TAAT motifsV$MYBL/VMYB.04 v-Myb, AMV v- 0.85 356-368 (+) 1.000 0.881 attAACGctggt180 myb c V$HOXF/CRX.01 Cone-rod (C) 0.94 366-382 (+) 1.000 0.962gtctTAATcacc 181 homeobox- aagcc containing transcriptionfactor/otx-like homeobox gene V$RCAT/CLTR_CA Mammalian C- 0.71 375-399(+) 1.000 0.718 aCCAAgccaag 182 AT.01 type LTR ctccttaaactgct CCAAT boxV$ETSF/ETS1.01 c-Ets-1 binding 0.92 409-425 (−) 1.000 0.921 actaggaGGAA183 site gcctag V$SF1F/FTF.01 Alpha (1)- 0.94 426-438 (−) 1.000 0.940gggcCAAGgtg 184 fetoprotein ac transcription factor (FTF),liver receptor homologue-1 (LRH-1) V$BCL6/BCL6.02 POZ/zinc finger 0.77436-452 (+) 1.000 0.785 cccctctTAGAa 185 protein, gccaa transcriptionalrepressor, translocations observed in diffuse large cell lymphomaV$HOXF/GSH2.01 Homeodomain 0.95 443-459 (−) 1.000 1.000 ggccTAATtgg 186transcription cttcta factor Gsh-2 V$CAAT/CAAT.01 Cellular and viral 0.90445-459 (+) 1.000 0.949 gaagCCAAtta 187 CCAAT box ggcc V$NKXH/NKX25.0Homeo domain 0.88 446-460 (−) 1.000 0.938 gggccTAATtg 188 2 factor Nkx-gctt 2.5/Csx, tinman homolog low affinity sites V$HOMF/S8.01Binding site for 0.97 448-460 (−) 1.000 0.999 gggccTAATtg 189 S8 type gchomeodomains V$HOXF/CRX.01 Cone-rod (D) 0.94 470-486 (−) 1.000 0.985atatTAATcccc 190 homeobox- gctgc containing transcriptionfactor/otx-like homeobox gene V$MZF1/MZF1.01 Myeloid zinc 0.99 473-481(+) 1.000 0.991 gcGGGGatt 191 finger protein MZF1 V$OCTB/TST1.01POU-factor Tst- 0.90 475-487 (+) 1.000 0.947 ggggATTAatat 192 1/Oct-6 gV$CART/CART1.01 Cart-1 (cartilage 0.86 477-493 (−) 1.000 0.926caTAATcatatt 193 homeoprotein 1) aatcc V$CART/CART1.01 Cart-1 (cartilage0.86 479-495 (+) 1.000 0.914 atTAATatgatta 194 homeoprotein 1) tgaaV$SATB/SATB1.01 Special AT-rich 0.94 479-493 (+) 1.000 0.957attAATAtgatta 195 sequence-binding tg protein 1, predominantlyexpressed in thymocytes, binds to matrix attachment regions (MARs)V$PDX1/PDX1.01 Pdx1 0.74 480-500 (+) 0.826 0.775 ttaatatgaTTAT 196(IDX1/IPF1) gaacaccc pancreatic and intestinal homeodomain TFV$GLIF/ZIC2.01 Zinc finger 0.89 491-505 (+) 1.000 0.932 atgaacaCCCCc 197transcription aat factor, Zic family member 2 (odd- paired homolog,Drosophila) V$CAAT/ACAAT.0 Avian C-type 0.83 497-511 (+) 1.000 0.905acccCCAAtctc 198 1 LTR CCAAT cca box V$RREB/RREB1.01 Ras-responsive 0.80499-513 (+) 1.000 0.841 cCCCAatctccc 199 element binding aga protein 1V$NRSF/NRSF.01 Neuron- 0.69 500-520 (−) 1.000 0.696 atcAGCAtctgg 200restrictive gagattggg silencer factor V$IKRS/LYF1.01 LyF-1 (Ikaros 1),0.98 502-514 (−) 1.000 1.000 atcTGGGagatt 201 enriched in B and gT lymphocytes V$AP4R/TAL1 ALP Tal-1 alpha/E47 0.87 505-521 (+) 1.0000.905 tctccCAGAtgc 202 HAE47.01 heterodimer tgatt V$RP58/RP58.01Zinc finger 0.84 507-519 (−) 1.000 0.865 tcagCATCtggg 203 protein RP58 a(ZNF238), associated preferentially with heterochromatin V$AP1R/NFE2.01NF-E2 p45 0.85 508-532 (+) 1.000 0.904 cccagatgCTG 204 Attcagccaggag cV$AP1R/NFL2.01 NF-E2 p45 0.85 508-532 (−) 1.000 0.882 gctcctggCTGA 205atcagcatctggg V$BEL1/BEL1.01 Bel-1 similar 0.81 510-532 (−) 1.000 0.818gctcctggctgaaT 206 region (defined CAGcatctg in Lentivirus LTRs)V$NRLF/NRL.01 Neural retinal 0.85 511-529 (−) 1.000 0.991 cctggCTGAatc207 basic leucine agcatct zipper factor (bZIP) V$AP1F/AP1.03Activator protein 0.94 515-525 (+) 0.885 0.970 gcTGATtcagc 208 1V$AP1F/AP1.03 Activator protein 0.94 515-525 (−) 0.857 0.963 gcTGAAtcagc209 1 V$HOXF/PTX1.01 Pituitary 0.94 523-539 (−) 1.000 0.944 ctcCTAAgctcc210 Homeobox 1 tggct (Ptx1, Pitx-1) V$ZBPF/ZNF219.01 Kruppel-like zinc0.91 528-550 (−) 1.000 0.926 gtgacctCCCCc 211 finger protein tcctaagctcc219 V$RXRF/VDR_RX VDR/RXR 0.85 531-555 (+) 1.000 0.889 gcttaggaggggG 212R.01 Vitamin D AGGtcactttat receptor RXR heterodimer siteV$ZBPF/ZBP89.01 Zinc finger 0.93 531-553 (−) 1.000 0.958 aaagtgacctCC213 transcription CCctcctaagc factor ZBP-89 V$EKLF/KKLF.01Kidney-enriched 0.91 534-550 (+) 1.000 0.913 taggagGGGGa 214kruppel-like ggtcac factor, KLF15 V$GLIF/ZIC2.01 Zinc finger 0.89536-550 (−) 1.000 0.945 gtgacctCCCCc 215 transcription tccfactor, Zic family member 2 (odd- paired homolog, Drosophila)V$RORA/TR2.01 Nuclear hormone 0.92 538-556 (+) 1.000 0.950 agggggaGGTC216 receptor TR2, actttata half site V$TBPF/TATA.01 Cellular and viral0.90 543-559 (−) 1.000 0.915 ccttaTAAAgtg 217 TATA box acctc elementsV$SRFF/SRF.01 Serum response 0.66 545-563 (−) 1.000 0.722 agaccctTATAa218 factor agtgacc V$SRFF/SRF.01 Serum response 0.66 546-564 (+) 1.0000.712 gtcacttTATAa 219 factor gggtctg V$TBPF/LTATA.01 Lentivirus LTR0.82 550-566 (+) 1.000 0.829 cttTATAagggt 220 TATA box ctgggV$MOKF/MOK2.01 Ribonucleopro- 0.74 552-572 (−) 1.000 0.772 gacccccccagac221 tein associated CCTTataa zinc finger protein MOK-2 (mouse)V$ZBPF/ZNF219.01 Kruppel-like zinc 0.91 553-575 (−) 1.000 0.948tctgaccCCCCc 222 finger protein agacccttata 219 V$GLIF/ZIC2.01Zinc finger 0.89 560-574 (−) 1.000 0.967 ctgacccCCCCa 223 transcriptiongac factor, Zic family member 2 (odd- paired homolog, Drosophila)V$MAZF/MAZR.01 MYC-associated 0.88 561-573 (+) 1.000 0.919 tctgggGGGGtc224 zinc finger a protein related transcription factor V$ZNFP/SZF1.01SZF1, 0.82 579-603 (−) 0.801 0.829 tcaGGGCtccag 225 hematopoieticctggatgactctg progenitor- restricted KRAB- zinc finger proteinV$AP4R/AP4.01 Activator protein 0.85 584- (+) 1.000 0.916 tcatcCAGCtgg226 4 600UTR agccc V$EBOX/ATF6.01 Member of b-zip 0.93 596-610 (−) 1.0000.970 cagCCACtcag 227 family, induced ggct by ER damage/stress,binds to the ERSE in association with NF-Y V$CAAT/CAAT.01Cellular and viral 0.90 597-611 (−) 0.826 0.937 tcagCCACtcag 228CCAAT box ggc V$HEAT/HSF1.01 Heat shock factor 0.84 621-645 (−) 1.0000.857 tgctcccacccaA 229 1 GAAtgctgcgaa V$OAZF/ROAZ.01 Rat C2H2 Zn 0.73625-641 (+) 0.750 0.779 caGCATtcttgg 230 finger protein gtggginvolved in olfactory neuronal differentiation V$OAZF/ROAZ.01Rat C2H2 Zn 0.73 626- (−) 0.750 0.744 tcCCACccaag 231 finger protein642nc aatgct involved in olfactory neuronal differentiationV$EGRF/EGR2.01 Egr-2/Krox-20 0.79 631- (+) 0.766 0.828 tcttGGGTggga 232early growth 647UTR gcagc response gene product V$EGRF/WT1.01Wilms Tumor 0.92 633-649 (+) 1.000 0.930 ttgggTGGGag 233 Suppressorcagcca V$RBPF/RBPJK.01 Mammalian 0.84 634- (+) 1.000 0.847 tgggTGGGagc234 transcriptional 648UTR agcc repressor RBP- Jkappa/CBF1V$OAZF/ROAZ.01 Rat C2H2 Zn 0.73 641-657 (+) 0.750 0.818 gaGCAGccacg 235finger protein ggtcag involved in olfactory neuronal differentiationV$EBOX/USF.03 Upstream 0.89 643-657 (−) 1.000 0.904 ctgaccCGTGg 236stimulating factor ctgc V$EBOX/MYCMA MYC-MAX 0.91 644-658 (+) 0.8420.919 cagccaCGGGt 237 X.03 binding sites cagc V$PAX5/PAX5.03 PAXS paired0.80 659- (+) 0.894 0.833 cacaagggCCA 238 domain protein 687genCagccatgaatgg cacag V$CLOX/CDPCR3. Cut-like 0.73 665- (+) 1.000 0.735ggccacagccatg 239 01 homeodomain 683gen aATGGc protein V$PAX5/PAX5.03PAX5 paired 0.80 674- (+) 1.000 0.800 catgaatgGCAC 240 domain protein702gen agaagtcctgagcc cca V$ZNFP/ZBRK1.01 Transcription 0.77 680-704 (−)0.813 0.847 catggggcTCA 241 factor with 8 Ggacttctgtgcca central zincfingers and an N- terminal KRAB domain V$PBXC/PBX1_ME Binding site for a0.77 699-715 (−) 0.750 0.860 attcTGTTtgaca 242 IS1.02 Pbx1/Meis1 tgggheterodimer V$TALE/TGIF.01 TG-interacting 1.00 700- (+) 1.000 1.000ccatGTCAaac 243 factor belonging 710nc to TALE class of homeodomainfactors V$SNAP/PSE.02 Proximal 0.73 745-763 (+) 1.000 0.734 gtgccCCTAatc244 sequence element ctcaact (PSE) of RNA polymerase III-transcribed genes V$HOXF/CRX.01 Cone-rod (E) 0.94 748-764 (+) 1.0000.965 ccccTAATcctc 245 homeobox- aacta containing transcriptionfactor/otx-like homeobox gene V$FAST/FAST1.01 FAST-1 SMAD 0.81 749-763(−) 0.983 0.829 agttgagGATTa 246 interacting ggg protein V$NR2F/HPF1.01HepG2-specific 0.78 767-787 (+) 0.750 0.801 ctaggcgcAAA 247P450 2C factor-1 Ttccaatcct V$SORY/HMGIY.0 HMGI(Y) high- 0.92 770-782(−) 1.000 0.938 tggAATTtgcgc 248 1 mobility-group c protein I (Y),architectural transcription factor organizing the framework ofa nuclear protein- DNA transcriptional complex V$HMTB/MTBF.01Muscle-specific 0.90 774-782 (−) 1.000 0.953 tggaATTTg 249Mt binding site V$LEFF/LEF1.01 TCF/LEF-1, 0.86 783-799 (−) 1.000 0.889actagacCAAA 250 involved in the gaggat Wnt signal transduction pathwayV$NRSF/NRSE.01 Neural- 0.67 794- (+) 0.782 0.762 tctagtacccCGG 251restrictive- 814nc Gggcagcc silencer-element V$ZBPF/ZF9.01Core promoter- 0.87 803- (+) 0.769 0.878 ccgggggCAG 252 binding protein825nc Ccccctctaacct (CPBP) with 3 Krueppel-type zinc fingersV$HICF/HIC1.01 Hypermethylated 0.93 804-816 (−) 1.000 0.970 ggggcTGCCcc253 in cancer 1, cg transcriptional repressor containing fiveKrüppel-like C2H2 zinc fingers, for optimal binding multiple bindingsites are required. V$NFKB/NFKAPP NF-kappaB (p50) 0.83 806-818 (−) 0.7500.865 aggGGGCtgcc 254 AB50.01 cc V$STAF/ZNF76_14 ZNF143 is the 0.76810-832 (+) 0.809 0.761 cagcCCCCtcta 255 3.01 human ortholog accttgggcctof Xenopus Staf, ZNF76 is a DNA binding protein related toZNF143 and Staf V$SF1F/SF1.01 SF1 0.95 819-831 (−) 1.000 0.966ggccCAAGgtt 256 steroidogenic ag factor 1 V$RXRF/VDR_RXBipartite binding 0.75 825- (+) 0.812 0.787 ttgggcctcagcag 257 R.06site of 849nc cAGGGgaggc VDR/RXR c heterodimers: 4 spacer nucleotidesbetween the two directly repeated motifs V$MYOD/MYF5.01 Myf5 myogenic0.90 831- (+) 1.000 0.903 ctcagCAGCag 258 bHLH protein 847nc gggaggV$NRSF/NRSF.01 Neuron- 0.69 831- (+) 1.000 0.705 ctcAGCAgcag 259restrictive 851nc? gggaggccac silencer factor V$ZBPF/ZF9.01Core promoter- 0.87 832-854 (−) 0.820 0.890 ggggtggCCTC 260binding protein ccctgctgctga (CPBP) with 3 Krueppel-type zinc fingersV$ZBPF/ZF9.01 Core promoter- 0.87 841- (+) 0.923 0.937 ggggaggCCA 261binding protein 863nc Cccctacaccttc (CPBP) with 3 Krueppel-typezinc fingers V$PLAG/PLAG1.01 Pleomorphic 0.88 847-867 (−) 0.958 0.929GGGGgaaggtg 262 adenoma gene taggggtggc (PLAG) 1, a developmentallyregulated C2H2 zinc finger protein V$ZBPF/ZNF202.01 Transcriptional 0.73859-881 (+) 1.000 0.776 ccttccCCCAgc 263 repressor, binds cacagccatccto elements found predominantly in genes that participate inlipid metabolism V$INSM/INSM1.01 Zinc finger 0.90 860-872 (−) 1.0000.965 tggctGGGGga 264 protein ag insulinoma- associated 1 (IA-1) functions as a transcriptional repressor V$MZF1/MZF1.02 Myeloid zinc0.99 860-868 (−) 1.000 0.994 tgGGGGaag 265 finger protein MZF1V$HAML/AML3.01 Runt-related 0.84 863-877 (−) 1.000 0.845 ggctGTGGctg 266transcription gggg factor 2/CBFA1 (core-binding factor, runtdomain, alpha subunit 1) V$NRF1/NRF1.01 Nuclear 0.78 889- (−) 0.7500.828 tctGCACaggc 267 respiratory factor 905nc gctgct 1 (NRF1), bZIPtranscription factor that acts on nuclear genes encoding mitochondrialproteins V$NRF1/NRF1.01 Nuclear 0.78 890- (+) 1.000 0.801 gcaGCGCctgt268 respiratory factor 906nc gcagaa 1 (NRF1), bZIP transcriptionfactor that acts on nuclear genes encoding mitochondrial proteinsV$SORY/HBP1.01 HMG box- 0.86 898- (+) 1.000 0.862 tgtgcagAATG 269containing 910nc aa protein 1 V$BRNF/BRN2.03 Brn-2, POU-III 0.92 923-941(+) 1.000 0.932 ggctccttaATT 270 protein class Ttttttt V$NKXH/NKX25.0Homeo domain 0.88 925-939 (+) 1.000 0.956 ctcctTAATttttt 271 2factor Nkx- t 2.5/Csx, tinman homolog low affinity sites V$CDXF/CDX1.01Intestine specific 0.94 939-957 (+) 1.000 0.948 tttttttTTTAaga 272homeodomain aataa factor CDX-1 V$HOXF/HOXB9.0 Abd-B-like 0.88 940-956(−) 1.000 0.888 tatttctTAAAaa 273 1 homeodomain aaaa protein Hoxb-9V$CEBP/CEBPB.01 CCAAT/enhancer 0.94 943- (+) 1.000 0.942 ttttttaaGAAAt274 binding protein 957nc aa beta V$HNF1/HNF1.01 Hepatic nuclear 0.80947-963 (−) 0.790 0.824 cATTAattatttct 275 factor 1 taa V$HOXF/BARX2.0Barx2, 0.95 948-964 (−) 1.000 0.967 tcatTAATtatttc 276 1 homeobox ttatranscription factor that preferentially binds to paired TAAT motifsV$BRNF/BRN3.01 Brn-3, POU-IV 0.78 949-967 (−) 1.000 0.990 gcctcattaATT277 protein class Atttctt V$BRNF/BRN4.01 POU domain 0.89 949- (+) 1.0000.894 aagaaataatTA 278 transcription 967nc ATgaggc factor brain 4V$LHXF/LMX1B.0 LIM- 0.91 949- (+) 1.000 0.962 aagaaaTAATta 279 1homeodomain 963nc atg transcription factor V$HOMF/S8.01 Binding site for0.97 950- (+) 1.000 0.997 agaaaTAATtaa 280 S8 type 962nc t homeodomainsV$HOXF/GSH1.01 Homeobox 0.85 952- (+) 1.000 0.863 aaataatTAATg 281transcription 968nc aggct factor Gsh-1 V$LHXF/LMX1B.0 LIM- 0.91 952-966(−) 1.000 0.946 cctcatTAATtat 282 1 homeodomain tt transcription factorV$RBIT/BRIGHT.0 Bright, B cell 0.92 952- (+) 1.000 0.961 aaataATTAatg283 1 regulator of IgH 964nc a transcription V$HOMF/S8.01Binding site for 0.97 953-965 (−) 1.000 0.992 ctcatTAATtatt 284 S8 typehomeodomains V$LHXF/LHX3.01 Homeodomain 0.81 953- (+) 1.000 0.851aataaTTAAtga 285 binding site in 967nc ggc LIM/Homeodo- main factor LHX3V$SORY/HBP1.01 HMG box- 0.86 953- (+) 1.000 0.876 aataattAATGa 286containing 965nc g protein 1 V$HOXF/BARX2.0 Barx2, 0.95 955- (+) 1.0000.987 taatTAATgagg 287 1 homeobox 971nc ctcct transcription factor thatpreferentially binds to paired TAAT motifs V$RXRF/VDR_RX VDR/RXR 0.86960-984 (−) 1.000 0.871 tcccaggtgagtG 288 R.02 Vitamin D AGGagcctcattreceptor RXR heterodimer site V$AP1F/AP1.03 Activator protein 0.94969-979 (−) 1.000 0.940 ggTGAGtgagg 289 1 V$AREB/AREB6.01 AREB6 (Atp1a10.93 972- (+) 1.000 0.933 cactcACCTgg 290 regulatory 984nc gaelement binding factor 6) V$PAX6/PAX6.02 PAX6 paired 0.89 973-991 (−)1.000 0.893 caggctgtcCCA 291 domain and Ggtgagt homeodomainare required for binding to this site V$AP2F/AP2.01 Activator protein0.90 1033- (−) 1.000 0.911 ctgGCCTtggg 292 2 1047 gaac V$EREF/ERR.01Estrogen related 0.87 1033- (+) 1.000 0.897 gttccccAAGG 293 receptor1051nc ccagcggg V$MZF1/MZF1.02 Myeloid zinc 0.99 1033- (−) 1.000 0.994ttGGGGaac 294 finger protein 1041 MZF1 V$SF1F/SF1.01 SF1 0.95 1035- (+)1.000 0.992 tcccCAAGgcc 295 steroidogenic 1047nc ag factor 1V$TEAF/TEF.01 Thyrotrophic 0.88 1044- (−) 0.968 0.894 ggcacaCATCc 296embryonic factor 1060 cgctgg V$SP1F/TIEG.01 TGFbeta- 0.83 1046- (+)0.750 0.878 agcGGGAtgtgt 297 inducible early 1060nc gcc gene (TIEG)/Early growth response gene alpha (EGRalpha) V$MAZF/MAZ.01 Myc associated0.90 1056- (−) 1.000 0.909 ggagGAGGgg 298 zinc finger 1068 cacprotein (MAZ) V$RXRF/VDR_RX Bipartite binding 0.74 1056- (−) 0.823 0.750gatgAGTTggg 299 R.03 site of 1080 aggaggaggggc VDR/RXR ac heterodimerswithout a spacer between directly repeated motifs V$EVI1/MEL1.02 MEL10.99 1071- (−) 1.000 0.997 cctgaaaGATG 300 (MDS1/EVI1- 1087 agttgglike gene 1) DNA-binding domain 2 V$HEAT/HSF1.01 Heat shock factor 0.841073- (−) 0.857 0.849 tcctcgtgttccTG 301 1 1097 AAagatgagttV$MYT1/MYT1L.0 Myelin 0.92 1073- (−) 0.818 0.927 tgaaAGATgag 302 1transcription 1085 tt factor 1-like, neuronal C2HC zinc finger factor 1V$STAT/STAT1.01 Signal transducer 0.77 1075- (−) 0.767 0.774cgtgttcctGAA 303 and activator of 1093 Agatgag transcription 1V$STAT/STAT.01 Signal 0.87 1077- (+) 1.000 0.911 catctttcaGGA 304transducers and 1095 Acacgag activators of transcription V$EBOX/NMYC.01N-Myc 0.92 1085- (−) 1.000 0.923 aatcctCGTGttc 305 1099 ctV$HEAT/HSF2.02 Heat shock factor 0.95 1089- (−) 1.000 0.967ttccagaaagcaA 306 2 1113 GAAtcctcgtgt V$HEAT/HSF1.01 Heat shock factor0.84 1097- (−) 1.000 0.874 ggacacttttccA 307 1 1121 GAAagcaagaatV$STAT/STAT1.01 Signal transducer 0.77 1099- (−) 0.767 0.798acttttccaGAA 308 and activator of 1117 Agcaaga transcription 1V$STAT/STAT.01 Signal 0.87 1101- (+) 1.000 0.895 ttgctttctGGAA 309transducers and 1119 aagtgt activators of transcription V$BCL6/BCL6.02POZ/zinc finger 0.77 1102- (+) 0.800 0.808 tgctttcTGGAa 310 protein,1118 aagtg transcriptional repressor, translocations observed indiffuse large cell lymphoma V$BNCF/BNC.01 Basonuclin, 0.85 1107- (+)1.000 0.852 tctggaaaagTG 311 cooperates with 1125 TCccagc USF1 in rDNAPo1I transcription) V$GATA/GATA2.0 GATA-binding 0.92 1127- (+) 1.0000.938 taggGATAagt 312 1 factor 2 1139 gt V$NKXH/NKX32.0 Homeodomain 0.961128- (+) 1.000 0.962 agggataAGTG 313 1 protein NKX3.2 1142 tcta (BAPX1,NKX3B, Bagpipe homolog) V$PAX1/PAX1.01 Pax1 paired 0.62 1135- (−) 0.7500.696 cCATTctgtgct 314 domain protein, 1153 agacact expressed in thedeveloping vertebral column of mouse embryos V$SORY/HBP1.01 HMG box-0.86 1142- (+) 1.000 0.860 agcacagAATG 315 containing 1154nc ggprotein 1 V$NKXH/NKX25.0 Homeo domain 0.88 1166- (+) 1.000 0.898gtgctTAATaaa 316 2 factor Nkx- 1180 tgc 2.5/Csx, tinman homolog lowaffinity sites V$HOXF/HOXC13. Homeodomain 0.91 1167- (+) 1.000 0.944tgcttaaTAAAt 317 01 transcription 1183 gctgg factor HOXC13 V$HOXC/HOX_PBHOX/PBX 0.81 1178- (+) 0.944 0.862 tgctGGATggat 318 X.01 binding sites1194 gcagg V$AIRE/AIRE.01 Autoimmune 0.86 1184- (+) 1.000 0.877atggatgcaggaa 319 regulator 1210 ggaaTGGAgga atg V$ETSF/ELF2.01Ets-family 0.90 1186- (+) 1.000 0.933 ggatgcaGGAA 320 member ELF-2 1202ggaatg (NERF1a) V$GKLF/GKLF.01 Gut-enriched 0.86 1191- (+) 0.779 0.864caggaaggaAT 321 Krueppel-like 1203 GG factor V$SORY/HBP1.01 HMG box-0.86 1192- (+) 1.000 0.904 aggaaggAATG 322 containing 1204 ga protein 1V$TEAF/TEF1.01 TEF-1 related 0.84 1192- (−) 1.000 0.859 ttcctcCATTcct323 muscle factor 1208 tcct V$ETSF/PU1.01 Pu.1 (Pu120) Ets- 0.89 1198-(+) 1.000 0.899 gaatggaGGAA 324 like transcription 1214 tgaatgfactor identified in lymphoid B- cells V$SORY/HBP1.01 HMG box- 0.861200- (+) 1.000 0.916 atggaggAATG 325 containing 1212 aa protein 1V$TEAF/TEF1.01 TEF-1 related 0.84 1200- (−) 1.000 0.884 cccattCATTcct326 muscle factor 1216 ccat V$SORY/HBP1.01 HMG box- 0.86 1204- (+) 1.0000.949 aggaatgAATG 327 containing 1216 gg protein 1 V$IRFF/IRF7.01Interferon 0.86 1208- (+) 0.936 0.885 atGAATgggaa 328 regulatory factor1226nc ggtctaga 7 (IRF-7) V$RBPF/RBPJK.02 Mammalian 0.94 1209- (+) 1.0000.942 tgaaTGGGaag 329 transcriptional 1223nc gtct repressor RBP-Jkappa/CBF1 V$IKRS/IK1.01 Ikaros 1, 0.92 1210- (+) 1.000 0.925gaatGGGAagg 330 potential 1222nc tc regulator of lymphocytedifferentiation V$RORA/NBRE.01 Monomers of the 0.89 1212- (+) 1.0000.947 atgggAAGGtct 331 nur subfamily of 1230nc agagcat nuclear receptors(nur77, nurr-1, nor-1) V$ZFIA/ZID.01 Zinc finger with 0.85 1225- (−)1.000 0.916 agGCTCcatgct 332 interaction 1237 c domain V$AIRE/AIRE.01Autoimmune 0.86 1238- (−) 0.916 0.863 atgtgggcgggtg 333 regulator 1264agcaTGGCttct ag V$EGRF/WT1.01 Wilms Tumor 0.92 1246- (−) 0.953 0.930gtgggCGGGtg 334 Suppressor 1262 agcatg V$SP1F/SP1.01 Stimulating 0.881250- (−) 1.000 0.907 atgtGGGCgggt 335 protein 1, 1264 gagubiquitous zinc finger transcription factor V$NKXH/HMX3.02 Hmx3/Nkx5-10.92 1258- (−) 1.000 0.933 ttaaTTAAatgtg 336 homeodomain 1272 ggtranscription factor V$CREB/E4BP4.01 E4BP4, bZIP 0.80 1259- (+) 0.7580.801 ccacatttaaTTA 337 domain, 1279 Acagctga transcriptional repressorV$BRNF/BRN3.02 Brn-3, POU-IV 0.89 1260- (−) 1.000 0.940 cagctgtTAATt 338protein class 1278 aaatgtg V$LHXF/LHX3.01 Homeodomain 0.81 1260- (+)1.000 0.944 cacatTTAAtta 339 binding site in 1274 aca LIM/Homeo-domain factor LHX3 V$OCT1/OCT1.05 Octamer-binding 0.89 1260- (+) 0.9000.942 caCATTtaattaa 340 factor 1 1274 ca V$HOMF/S8.01 Binding site for0.97 1261- (+) 1.000 0.997 acattTAATtaa 341 S8 type 1273 c homeodomainsV$HOXF/PHOX2.01 Phox2a (ARIX) 0.87 1262- (+) 1.000 0.877 cattTAATtaac342 and Phox2b 1278 agctg V$NKXH/NKX25.0 Homeo domain 0.88 1262- (−)1.000 0.898 gctgtTAATtaa 343 2 factor Nkx- 1276 atg 2.5/Csx, tinmanhomolog low affinity sites V$PBXC/PBX1_ME Binding site for a 0.77 1262-(+) 0.750 0.781 cattTAATtaac 344 IS1.02 Pbx1/Meis1 1278 agctgheterodimer V$RBIT/BRIGHT.0 Bright, B cell 0.92 1262- (−) 1.000 0.967tgttaATTAaatg 345 1 regulator of IgH 1274 transcription V$FAST/FAST1.01FAST-1 SMAD 0.81 1263- (−) 0.850 0.845 agctgttAATTa 346 interacting 1277aat protein V$LHXF/LHX3.01 Homeodomain 0.81 1263- (−) 1.000 0.870agctgTTAAtta 347 binding site in 1277 aat LIM/Homeo- domain factor LHX3V$RBIT/BRIGHT.0 Bright, B cell 0.92 1263- (+) 1.000 0.941 atttaATTAaca348 1 regulator of IgH 1275 g transcription V$ZNFP/SZF1.01 SZF1, 0.821263- (−) 0.875 0.866 tcaGGGActca 349 hematopoietic 1287 gctgttaattaaatprogenitor- restricted KRAB- zinc finger protein V$ATBF/ATBF1.01AT-binding 0.79 1264- (−) 1.000 0.812 ctcagctgttAAT 350 transcription1280 Taaa factor 1 V$HOMF/S8.01 Binding site for 0.97 1264- (−) 1.0000.997 gctgtTAATtaa 351 S8 type 1276 a homeodomains V$HEN1/HEN1.02 HEN10.81 1265- (−) 1.000 0.845 agggactcaGCT 352 1285 GttaattaaV$NKXH/HMX3.02 Hmx3/Nkx5-1 0.92 1265- (+) 1.000 0.927 ttaaTTAAcagc 353homeodomain 1279 tga transcription factor V$AP4R/AP4.02Activator protein 0.92 1267- (−) 1.000 0.950 ggactcAGCTgt 354 4 1283taatt V$AP1R/NFE2.01 NF-E2 p45 0.85 1268- (+) 1.000 0.865 attaacagCTGA355 1292 gtccctgatgtca V$BEL1/BEL1.01 Bel-1 similar 0.81 1270- (−) 1.0000.842 tgacatcagggac 356 region (defined 1292 TCAGctgtta in LentivirusLTRs) V$CREB/CREBP1.0 cAMP- 0.85 1278- (−) 1.000 0.851 taaggaTGACat 3571 responsive 1298 cagggactc element binding protein 1 V$CREB/ATF2.01Activating 0.87 1279- (+) 0.814 0.871 agtcccTGATgt 358 transcription1299 catccttac factor 2 V$E4FF/E4F.01 GLI-Krueppel- 0.82 1284- (+) 0.8420.824 ctgATGTcatcc 359 related 1296 t transcription factor, regulatorof adenovirus E4 promoter V$HOXF/PTX1.01 Pituitary 0.94 1299- (−) 1.0000.949 tttCTAAgctctt 360 Homeobox 1 1315 cgag (Ptx1, Pitx-1)V$TBPF/ATATA.01 Avian C-type 0.78 1302- (−) 1.000 0.781 ttgtttcTAAGct361 LTR TATA box 1318 cttc V$XBBF/RFX1.01 X-box binding 0.89 1302- (+)0.881 0.890 gaagagcttaGA 362 protein RFX1 1320 AAcaaag V$LEFF/LEF1.01TCF/LEF-1, 0.86 1309- (+) 1.000 0.884 ttagaaaCAAA 363 involved in the1325nc gagtgg Wnt signal transduction pathway V$RBPF/RBPJK.02 Mammalian0.94 1319- (+) 1.000 0.977 agagTGGGaaa 364 transcriptional 1333 nc tgctrepressor RBP- Jkappa/CBF1 V$CP2F/CP2.01 CP2 0.90 1331- (−) 1.000 0.932agCTGGgtaaa 365 1349 gctagagc V$SRFF/SRF.02 Serum response 0.84 1362-(+) 0.888 0.842 taaggCAAAttg 366 factor 1380 ggccatt V$CART/XVENT2.Xenopus 0.82 1366- (+) 0.750 0.882 gcAAATtgggc 367 01 homeodomain 1382cattaa factor Xvent-2; early BMP signaling response V$CART/XVENT2.Xenopus 0.82 1367- (−) 1.000 0.835 ttTAATggccca 368 01 homeodomain 1383atttg factor Xvent-2; early BMP signaling response V$PDX1/1SL1.01Pancreatic and 0.82 1370- (−) 1.000 0.875 ctgagctttTAAT 369intestinal lim- 1390 ggcccaat homeodomain factor V$NKXH/HMX3.02Hmx3/Nkx5-1 0.92 1372- (−) 1.000 0.946 gcttTTAAtggc 370 homeodomain 1386cca transcription factor V$HOXF/HOXC13. Homeodomain 0.91 1373- (+) 1.0000.932 gggccatTAAA 371 01 transcription 1389 agctca factor HOXC13V$NKXH/HMX3.02 Hmx3/Nkx5-1 0.92 1375- (+) 1.000 0.953 gccaTTAAaag 372homeodomain 1389 ctca transcription factor V$MYBL/VMYB.05v-Myb, variant of 0.90 1404- (+) 1.000 0.990 attAACGgtggt 373 AMV v-myb1416 g V$AHRR/AHRARN Aryl hydrocarbon/ 0.77 1423- (−) 0.750 0.781cctgtggataGA 374 T.02 Arnt 1447 GTgtgaaagcaa heterodimers, c fixed coreV$EVI1/EVI1.06 Ecotropic viral 0.83 1440- (+) 0.750 0.835 tccacaGGATa375 integration site 1 1456 gattga encoded factor, amino-terminalzinc finger domain V$HOXC/HOX_PB HOX/PBX 0.81 1442- (+) 0.944 0.814cacaGGATaga 376 X.01 binding sites 1458 ttgaaa V$HOXC/PBX1.01Homeo domain 0.78 1446- (+) 1.000 0.809 ggataGATTga 377 factor Pbx-11462 aactgc V$IRFF/ISRE.01 Interferon- 0.81 1447- (+) 1.000 0.829gatagattGAAA 378 stimulated 1465 ctgccag response element V$HOXH/MEIS1B_Meis1b and 0.78 1450- (−) 0.750 0.781 TGGCagtttcaat 379 HOXA9.01Hoxa9 form 1464 ct heterodimeric binding complexes on target DNAV$NR2F/ARP1.01 Apolipoprotein 0.82 1469- (−) 0.857 0.897 ccagggtcaggG380 AI regulatory 1489 ATCaggtgg protein 1, NR2F2 V$MEF3/MEF3.01MEF3 binding 0.89 1474- (−) 1.000 0.943 gggTCAGggat 381 site, present in1486 ca skeletal muscle- specific transcriptional enhancersV$RORA/TR4.01 Nuclear hormone 0.84 1474- (−) 1.000 0.841 atcccagGGTC 382receptor TR4 1492 agggatca homodimer binding site V$CSEN/DREAM.0Downstream 0.95 1476- (−) 1.000 0.974 ggGTCAgggat 383 1 regulatory 1486element- antagonist modulator, Ca2+-binding protein of the neuronalcalcium sensors family that binds DRE (downstream regulatoryelement) sites as a tetramer V$CP2F/CP2.01 CP2 0.90 1493- (+) 1.0000.969 ggCTGGattga 384 1511 gcaatgag V$HOXC/PBX1.01 Homeo domain 0.781493- (+) 1.000 0.811 ggctgGATTga 385 factor Pbx-1 1509 gcaatgV$CEBP/CEBPB.01 CCAAT/enhancer 0.94 1496- (+) 1.000 0.984 tggattgaGCAA386 binding protein 1510 tga beta V$CAAT/NFY.03 Nuclear factor Y 0.811513- (+) 1.000 0.873 agagCCAAgca 387 (Y-box binding 1527 gcac factor)V$STAF/ZNF76_14 ZNF143 is the 0.76 1522- (−) 1.000 0.765 tagcCCCAggg 3883.01 human ortholog 1544 gactctgtgctg of Xenopus Staf, ZNF76 is a DNAbinding protein related to ZNF143 and Staf V$NOLF/OLF1.01 Olfactory 0.821526- (+) 1.000 0.879 acagagTCCCct 389 neuron-specific 1548 ggggctagaggfactor V$AP2F/AP2.02 Activator protein 0.92 1531- (−) 0.905 0.941ctaGCCCcagg 390 2 alpha 1545 ggac V$ZBPF/ZNF202.01 Transcriptional 0.731536- (−) 0.761 0.739 gcctccTCCAcc 391 repressor, binds 1558 tctagccccagto elements found predominantly in genes that participate inlipid metabolism V$IKRS/IK1.01 Ikaros 1, 0.92 1561- (+) 1.000 0.933tcctGGGAatgg 392 potential 1573 g regulator of lymphocytedifferentiation V$TEAF/TEF1.01 TEF-1 related 0.84 1561- (−) 1.000 0.855ttttccCATTccc 393 muscle factor 1577 agga V$IRFF/IRF7.01 Interferon 0.861565- (+) 0.936 0.895 ggGAATggga 394 regulatory factor 1583nc aaaacccca7 (IRF-7) V$LTUP/TAACC.01 Lentiviral TATA 0.71 1565- (+) 1.000 0.721gggaatgggaaa 395 upstream element 1587nc AACCccaactt V$RBPF/RBPJK.02Mammalian 0.94 1566- (+) 1.000 0.947 ggaaTGGGaaa 396 transcriptional1580nc aacc repressor RBP- Jkappa/CBF1 V$IKRS/IK1.01 Ikaros 1, 0.921567- (+) 1.000 0.927 gaatGGGAaaa 397 potential 1579nc ac regulator oflymphocyte differentiation V$NFKB/CREL.01 c-Rel 0.91 1571- (−) 1.0000.971 tggggtttTTCCc 398 1583 V$CIZF/NMP4.01 NMP4 (nuclear 0.97 1572- (+)1.000 0.986 ggAAAAacccc 399 matrix protein 4)/ 1582nc CIZ (Cas-interacting zinc finger protein) V$SRFF/SRF.02 Serum response 0.84 1576-(−) 0.888 0.881 gacccCAAAgt 400 factor 1594 tggggttt V$MYT1/MYT1.02MyT1 zinc 0.88 1578- (−) 1.000 0.882 ccaAAGTtggg 401 finger 1590 gttranscription factor involved in primary neurogenesis Cartharius K, etal. (2005) Bioinformatics 21, 2933-42.

Example 4

Utilising the data from Examples 2 and 3, a suite of constructs aregenerated containing various shRNA suppressors and/or replacementrhodopsin nucleic acids enhanced with additional promoter sequences,known to be conserved between vertebrate species and various sequencesknown to enhance expression at RNA and/or protein levels. FIGS. 9 and 16represents diagrammatically sequences cloned in suppression and/orreplacement constructs. Notably, any combination of the elements andconserved regions outlined and indeed other elements that can modulategene expression could be used in the invention to control expression ofsuppression and/or replacement components.

Suppression and/or replacement constructs (FIG. 9) were then used togenerate recombinant AAV2/5 viruses using the procedures provided inExample 1. AAV2/5 suppression and/or replacement vectors were evaluatedin 129 wild type (WT) mice for levels of expression of suppressorsand/or replacement nucleic acids at the RNA and protein levels asdetailed in Example 1. FIG. 10A illustrates a comparison using an RNAseprotection assay of levels of human rhodopsin expression from the RHO-Mtransgene in RHO-M mice (lane M) versus the rhodopsin expressionobtained from the suppression and replacement constructs in rAAV2/5subretinally injected into wild type 129 mice (lanes B8, B9, B11, B12,B13, B16, B8). FIG. 10A illustrates that AAV-BBB, AAV-BB10, AAV-BB11,AAV-BB12, AAV-BB13 and AAV-BB16 express the human rhodopsin replacementgene in RNA extracted from 129 wild type mice subretinally injected withthese suppression and or replacement constructs. AAV-BB8, AAV-BB10 andAAV-BB11 express human rhodopsin at lower levels than AAV-BB12, AAV-BB13and AAV-BB16.

Further evaluation of suppression and replacement vectors wasundertaken. FIG. 11 provides a comparative analysis of human rhodopsinexpression from rAAV2/5 suppression and replacement vectors using realtime RT-PCR. FIG. 11 illustrates replacement rhodopsin expression levelsin RNA extracted from 129 wild type mice subretinally injected withsuppression and/or replacement constructs. Expression levels were alsodetermined in Rho-M transgenic mice which express a rhodopsinreplacement construct termed rCC and display normal retinal function.Suppression and replacement vectors AAV-BB12, AAV-BB13, AAV-BB16 andAAV-BB18 express approximately in the same order of magnitude as levelsof replacement rhodopsin transcript in Rho-M mice, indicating thatenhanced replacement constructs with enhancer elements and conservedregions may express sufficient levels of rhodopsin to sustain afunctional retina in vivo.

FIG. 12 illustrates retinal histology of adult wild type mouse retinassubretinally injected with 2 ul of 2×1012 particle/ml of differentsuppression and replacement rhodopsin AAV vectors (see FIG. 9). Twoweeks post-injection of AAV vectors transduced eyes were removed, fixedin 4% paraformaldehyde and cryosectioned (12 um). Subsequently, sectionswere stained with human specific anti-RHO antibody to visualiseexpression of replacement-RHO using Cy3 label (red) on the secondaryantibody; cell nuclei were counterstained with DAPI (blue). A: AAV-BBB,B: AAV-BB13, C: AAV-BB24, D: AAV-Q8, E: AAV-Q26, F: retina fromuninjected Rho-M transgenic mouse expressing RHO (positive control).Clear evidence of human rhodopsin expression from AAV suppression andreplacement vectors was obtained. Sections indicate different levels ofhuman RHO expression from the AAV suppression and replacement vectorsunder evaluation. OS: photoreceptor outer segments; IS: photoreceptorinner segments; ONL: outer nuclear layer; INL: inner nuclear layer; GCL:ganglion cell layer.

To explore efficacy of the suppression component of the suppression andreplacement approach delivered using AAV, a variety of suppression onlyvectors were generated with an EGFP reporter gene (see FIG. 9). AdultNHR transgenic mice on a rho−/− background, therefore expressing normalhuman RHO but not mouse rho, were transduced by subretinal injection of2 ul of 2×10¹² particle/ml of AAV-shQ1-EGFP (A) or AAV-shNT-EGFP (B).Two weeks after injection, eyes were removed, fixed in 4%paraformaldehyde and cryosectioned (FIG. 13). AAV-shQ1-EGFP expressesshRNA-Q1, which targets RHO, while AAV-shNT-EGFP expresses anon-targeting shRNA (see FIG. 9 for constructs). Both constructs expressEGFP allowing tracking of the transduced cell populations (green).Sections were counterstained with DAPI (blue) to label the position ofthe nuclear layers. A significant reduction in the photoreceptor cellnumber in the transduced part of the outer nuclear layer was apparent inthe AAV-shQ1-EGFP injected (A) retinas compared to those of injectedwith AAV-shNT-EGFP (B) (FIG. 13). IS: photoreceptor inner segments; ONL:outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer.

Adult RHO-347 transgenic mice carrying a dominant RHO mutation causingretinal degeneration akin to human RP, were subretinally injected with 2ul of 2×10¹² particle/ml of AAV-shNT (A) or AAV-shQ1 (B) vectors (FIG.14A). Two weeks post-injection transduced eyes were removed, fixed in 4%paraformaldehyde and cryosectioned (12 um). AAV-shQ1 expresses shRNA-Q1,which targets RHO, while AAV-shNT expresses a non-targeting shRNA. Bothconstructs express EGFP allowing tracking of the transduced part of theretina (green). Sections were counterstained with DAPI (blue) toindicate positions of the nuclear layers. A significant reduction of thephotoreceptor cell numbers in the transduced part of the outer nuclearlayer in the AAV-shNT injected or the uninjected (C) retinas wasapparent due to the degenerative effects of RHO-347 transgene (FIG.14A). A significantly preserved outer nuclear layer is detected in theAAV-shQ1 transduced retinas, where shRNA-Q1 effectively suppresses theRHO-347 transcript therefore reducing retinal degeneration (FIG. 14A).Note that the mouse rhodopsin gene (expressed in these retinas) wasrefractory to suppression by shRNA-Q1 due to the presence of nucleotidechanges at the target site for Q1 siRNA-based suppression. Suppressionof human rhodopsin and replacement using the degeneracy of the geneticcode provided therapeutic benefit at a histological level in RHO-347mice.

In addition, FIG. 14D provides evidence of an improvement in theelectroretinogram (ERG) in RHO-347 eyes treated with AAV-shQ1-EGFPversus eyes treated with AAV-shNT-EGFP. In FIG. 14D a representativemaximum ERG response of a RHO-347 mouse, containing a human rhodopsintransgene with a mutation at codon 347, subretinally injected withAAV2/5 constructs is presented. This RHO-347 mouse normally displays aphenotype similar to autosomal dominant RP. The top figure is theresponse of the right eye, which received an injection of AAV-shQ1-EGFP,a AAV2/5 vector containing suppressor siRNA Q1 driven by an H1 promoter(shQ1) and a CMV-driven EGFP gene. The left eye received anAAV-shNT-EGFP, a AAV2/5 containing a non-targeting (control) siRNAdriven by an H1 promoter (shNT) and a CMV-driven EGFP gene. As can beseen above, the maximum response is significantly greater in the treatedright eye than in the control left eye, indicating that suppression ofthe mutant rhodopsin transgene leads to some rescue at the ERG level.

Example 5 Sequences of Various Elements Designed to Enhance Expressionof Replacement Constructs

As described, enhancer elements, conserved regions A through I and/ortranscription factor binding sites and/or other regulatory elementsand/or epigenetic elements may be combined to improve expression ofreplacement constructs (see FIGS. 9 and 15 and Tables 1, 2, 9-13). Theseelements can be used in many different combinations to achieve optimumexpression, as demonstrated in the Examples provided above. Additionalexamples include inter alia a construct comprising a human rhodopsingene expressed from a composite promoter element containing the 484 bpmouse rhodopsin promoter together with the CMV enhancer, the rhodopsinpromoter enhancer element, the rhodopsin promoter conserved region B andflanked at the 3′end of the gene by a woodchuck posttranscriptionalregulatory element and a minimal poly A sequence. Another example issimilar to the one above but instead of the CMV enhancer, it containsmultiples of the CRX and/or NRL binding sites.

Example 6 Utilisation of Neuroprotective/Neurotrophic Factors inConjunction with Suppression and Replacement

As described above, there is evidence from the prior art thatneurotrophic/neuroprotective factors can improve cell viability and orcell functioning, the sequences encoding a number of these factors areprovided in FIG. 17. FIG. 18 provides suppression and replacementconstructs containing genetic elements that are beneficial for neuronalcell survival. In the example, the suppression and replacement constructpAAV-BB18 (FIG. 9) has been combined with the gene encoding theneurotrophic factor GDNF, driven by a small UCOE (chromatin openingelement. A Thrasher, Abstract 36, British Society for Gene Therapy 5thAnnual Conference 2008) promoter. Notably other neurotrophic factors andor genes encoding neurotrophic factors such as, for example, Neurturinmay also be used in combination with any of the suppression andreplacement constructs described. In example A (FIG. 18), the additionalelement, in this case sequence encoding GDNF is co-located with thesuppression and replacement construct within the two AAV invertedterminal repeat sequences, ITS1 and ITS2. In the second example, B (FIG.18), the GDNF gene and its promoter are not co-located with thesuppression and replacement elements within ITS1 and ITS2, but arelocated within the backbone of the plasmid used to generate AAV. Since asmall proportion of the backbone is packaged during AAV production, thisresults in a mixed population of AAVs with the majority containing thesuppression and replacement elements and a minority the GDNF elements.

AAV vectors generated to contain suppression, replacement andneurotrophic/neuroprotection components can be subretinally injectedinto wild type mice and or into mice with inherited retinaldegenerations such as the RHO-347 and Pro23H is mice described in theExamples above.

TABLE 14 Enhancers CMV Enhancer (SEQ ID NO: 402)CCGCGTTACA TAACTTACGG TAAATGGCCC GCCTGGCTG ACCGCCCAACG ACCCCCGCCC ATTGACGTCA ATAATGACGTATGTTCCCAT AGTAACGCCA ATAGGGACTT TCCATTGACGTCAATGGGTG GAGTATTTAC GGTAAACTGC CCACTTGGCAGTACATCAAG TGTATCATAT GCCAAGTACG CCCCCTATTGACGTCAATGA CGGTAAATGG CCCGCCTGGC ATTATGCCCAGTACATGACC TTATGGGACT TTCCTACTTG GCAGTACATCTACGTATTAG TCATCGCTAT TACCATGGTG ATGCGGTTTTGGCAGTACAT CAATGGGCGT GGATAGCGGT TTGACTCACGGGGATTTCCA AGTCTCCACC CCATTGACGT CAATGGGAGTTTGTTTTGGC ACCAAAATCA ACGGGAC Rhodopsin promoter conserved REGION A(SEQ ID NO: 403) GAGTGTCTAATTGCTTATGATCATGCATGCTCTCTCTCCCACTAAACATTTATTAATGTGTTAGGATTTCCATTAGCGCGTGCCTTGAACTGAAATCATTTGCATATGGCTGGGAAAAAGTGGGGTGAGGGAGGAAACAGTGCCAGCTCCCCAACAGGCGTCAATCACAGTGACAGATCAGATGG Rhodopsin Promoter Enhancer Element(contains Crx D(−) & CrxE (+) & NRL binding sites) (SEQ ID NO: 404TTTCTGCAGCGGGGATTAATATGATTATGAACACCCCCAATCTCCCAGATGCTGATTCAGCCAGGAGGTACC Crx D(−) (SEQ ID NO: 405)GCGGGGATTAATAT CrxE (+)(SEQ ID NO: 406) TGAACACCCCCAATCTCNRL (SEQ ID NO: 407) TGCTGATTCAGC Rhodopsin promoter conserved region B(SEQ ID NO: 408) TCTGCTGACCCAGCAACACTCTTTCCTTCTGAGGCTTAAGAGCTATTAGCGTAGGTGACTCAGTCCCTAATCCTCC Human rhodopsin polyA region F (SEQ ID NO: 409) GACCTGCCTAGGACTCTGTGGCCGACTATAGGCGTCTCCCATCCCCTACACCTTCCCCCAGCCACAGCCATCCCACCAGGAGCAGCGCCTGTGCAGAATGAACGAAGTCACATAGGCTCCTTAATTTTTTTTTTTTTTTTAAGAAATAAT TAATGAGGCTCCTCACTCHuman rhodopsin polyA region G  (SEQ ID NO: 410)ACCTGGGACAGCCTGAGAAGGGACATCCACCAAGACCTACTGATCTGGAGTCCCACGTTCCCCAAGGCCAGCGGGATGTGTGCCCCTCCTCCTCCCAACTCATCTTTCAGGAACACGAGGATTCTTGCTTTCTGGAAAAGTGTCCCAGCTTAGGGATAAGTGTCTAGCACAGAATGGGGCACACAGTAGGTGCTTAATAAATGCTGGATGGATGCAGGAAGGAATGGAGGAATGAATGGG AAGGGAGAACATAGGATCCSV40 Minimal polyA (SEQ ID NO: 411)AATAAAGGAAATTTATTTTCATGCAATAGTGTGTTGGTTTTTTGTGTGWPRE from pSK11 (SEQ ID NO: 412) GGATCC AATCAACCTCTGGATTACAA AATTTGTGAA AGATTGACTG GTATTCTTAACTATGTTGCT CCTTTTACGC TATGTGGATA CGCTGCTTTAATGCCTTTGT ATCATGCTAT TGCTTCCCGT ATGGCTTTCATTTTCTCCTC CTTGTATAAA TCCTGGTTGC TGTCTCTTTATGAGGAGTTG TGGCCCGTTG TCAGGCAACG TGGCGTGGTGTGCACTGTGT TTGCTGACGC AACCCCCACT GGTTGGGGCATTGCCACCAC CTGTCAGCTC CTTTCCGGGA CTTTCGCTTTCCCCCTCCCT ATTGCCACGG CGGAACTCAT CGCCGCCTGCCTTGCCCGCT GCTGGACAGG GGCTCGGCTG TTGGGCACTGACAATTCCGT GGTGTTGTCG GGGAAGCTGA CGTCCTTTCCATGGCTGCTC GCCTGTGTTG CCACCTGGAT TCTGCGCGGGACGTCCTTCT GCTACGTCCC TTCGGCCCTC AATCCAGCGGACCTTCCTTC CCGCGGCCTG CTGCCGGCTC TGCGGCCTCTTCCGCGTCTT CGCCTTCGCC CTCAGACGAG TCGGATCTCC CTTTGGGCCG CCTCCCCWPRE from pSin11 (SEQ ID NO: 413) GAGCAT CTTACCGCCATTTATTCCCA TATTTGTTCT GTTTTTCTTG ATTTGGGTATACATTTAAAT GTTAATAAAA CAAAATGGTG GGGCAATCATTTACATTTTT AGGGATATGT AATTACTAGT TCAGGTGTATTGCCACAAGA CAAACATGTT AAGAAACTTT CCCGTTATTTACGCTCTGTT CCTGTTAATC AACCTCTGGA TTACAAAATTTGTGAAAGAT TGACTGATAT TCTTAACTAT GTTGCTCCTTTTACGCTGTG TGGATATGCT GCTTTATAGC CTCTGTATCTAGCTATTGCT TCCCGTACGG CTTTCGTTTT CTCCTCCTTGTATAAATCCT GGTTGCTGTC TCTTTTAGAG GAGTTGTGGCCCGTTGTCCG TCAACGTGGC GTGGTGTGCT CTGTGTTTGCTGACGCAACC CCCACTGGCT GGGGCATTGC CACCACCTGTCAACTCCTTT CTGGGACTTT CGCTTTCCCC CTCCCGATCGCCACGGCAGA ACTCATCGCC GCCTGCCTTG CCCGCTGCTGGACAGGGGCT AGGTTGCTGG GCACTGATAA TTCCGTGGTG TTGTC

INCORPORATION BY REFERENCE

The contents of all cited references (including literature references,patents, patent applications, and websites) that maybe cited throughoutthis application are hereby expressly incorporated by reference. Thepractice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, which are wellknown in the art.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting of the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are therefore intended to be embracedherein.

1. A vector for expression of a suppression agent for a disease causinggene and/or a replacement nucleic acid that is not recognized by thesuppression agent, wherein the vector comprises at least one of theconserved regions selected from: conserved region B from the rhodopsingene represented by SEQ ID NO: 93, or a variant or equivalent thereof;conserved region C from the rhodopsin gene represented by SEQ ID NO: 94,or a variant or equivalent thereof; conserved region F and G from therhodopsin gene represented by SEQ ID NO: 97; and conserved region A fromthe rhodopsin gene represented by SEQ ID NO:
 92. 2. The vector accordingto claim 1, wherein the vector additionally comprises: (i) conservedregion D from the rhodopsin gene represented by SEQ ID NO: 95, orvariant or equivalent thereof; and/or (ii) at least one of conservedregions H and I from the rhodopsin gene represented by SEQ ID NOs: 98and 99 respectively, or variants or equivalents thereof.
 3. The vectoraccording to claim 1, wherein the vector comprises at least one of eachof conserved regions B, C, D, E, F and G, H, I and A, from the rhodopsingene represented by SEQ ID NOs: 93, 94, 95, 96, 97, 98, 99 and 92, orvariants or equivalents thereof.
 4. The vector according to claim 1,wherein the vector is an AAV vector.
 5. The vector according to claim 1,wherein the vector comprises at least one regulatory element selectedfrom the group consisting of a stuffer, an insulator, a silencer, anintron sequence, a post translational regulatory element, atranscription factor binding site, and an enhancer.
 6. The vector ofclaim 5, wherein said regulatory element(s) is: (i) an enhancer selectedfrom the group consisting of SEQ ID NOs: 87-89, or a variant orequivalent thereof; or (ii) an enhancer selected from the groupconsisting of SEQ ID NOs: 402-413, or a variant or equivalent thereof.7. The vector according to claim 5, wherein the vector comprises atleast one transcription factor binding site sequence selected from thegroup consisting of SEQ ID NOs: 100-401, or variant or equivalentthereof.
 8. The vector according to claim 1, wherein the vectorcomprises a chromatin opening element and/or a sequence encoding aneurotrophic or neuroprotective factor.
 9. The vector according to claim1, wherein the vector comprises at least one suppression agent and/or atleast one replacement nucleic acid.
 10. The vector according to claim 1,wherein said disease is a disease of the eye.
 11. The vector accordingto claim 1, wherein the replacement nucleic acid encodes a rhodopsingene.
 12. A suppression agent for use in the vector according to claim1, wherein said suppression agent comprises: (i) a nucleotide sequenceselected from the group consisting of SEQ ID NOs: 75, 77, 79, 81, 83,85, 414, 415, 416, 417, 418, 419, 420 and 421, or variant or equivalentthereof; (ii) a nucleotide sequence selected from the group consistingof SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,31, and 33, or variant or equivalent thereof; or (iii) a nucleotidesequence selected from the group consisting of SEQ ID NOs: 35-67, orvariant or equivalent thereof.
 13. A replacement nucleic acid for use inthe vector according to claim 1, wherein said replacement nucleic acidcomprises: (i) a nucleotide sequence selected from the group consistingof SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, and 68, or variant or equivalent thereof; or (ii) a nucleotidesequence selected from the group consisting of SEQ ID NOs: 76, 78, 80,82, 84, and 86, or variant or equivalent thereof.
 14. The vectoraccording to claim 1, wherein said vector comprises at least onesuppression agent, wherein said suppression agent comprises: (i) anucleotide sequence selected from the group consisting of SEQ ID NOs:75, 77, 79, 81, 83, 85, 414, 415, 416, 417, 418, 419, 420 and 421, orvariant or equivalent thereof; (ii) a nucleotide sequence selected fromthe group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, and 33, or variant or equivalent thereof; or(iii) a nucleotide sequence selected from the group consisting of SEQ IDNOs: 35-67, or variant or equivalent thereof.
 15. The vector accordingto claim 1, wherein said vector comprises at least one replacementnucleic acid, wherein said replacement nucleic acid comprises: (i) anucleotide sequence selected from the group consisting of SEQ ID NOs: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 68, orvariant or equivalent thereof; or (ii) a nucleotide sequence selectedfrom the group consisting of SEQ ID NOs: 76, 78, 80, 82, 84, and 86, orvariant or equivalent thereof.
 16. The vector according to claim 14,wherein said vector comprises at least one replacement nucleic acid,wherein said replacement nucleic acid comprises: (i) a nucleotidesequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 68, or variantor equivalent thereof; or (ii) a nucleotide sequence selected from thegroup consisting of SEQ ID NOs: 76, 78, 80, 82, 84, and 86, or variantor equivalent thereof.
 17. A therapeutic composition comprising at leastone vector according to claim
 1. 18. A cell comprising the vector ofclaim
 1. 19. A transgenic mouse comprising the vector of claim
 1. 20. Amethod of suppressing the expression of a mutant gene and replacingexpression of the mutant gene with a replacement nucleic acid, themethod comprising the steps of administering to a mammal the therapeuticcomposition of claim
 17. 21. A method of treating ocular disease, themethod comprising the step of administering the vector of claim
 1. 22. Amethod of treating ocular disease, the method comprising the step ofadministering the suppression agent according to claim
 12. 23. A methodof treating ocular disease, the method comprising the step ofadministering the replacement nucleic acid according to 13.